Functional Analysis of the Histidine N-Methyltransferase SETD3 in Endometriosis

Examination of SETD3 gene expression in endometriosis patients’ tissue and controls was performed via Turku Endometriosis database EndometDB ( https://endometdb.utu.fi/ , accessed on 30 October 2022). This web-based database incorporates the expression data from 115 patients and 53 controls as described previously [ 21 ]. All clinical and sample data were selected for analysis and SETD3 expression data was presented in boxplots on a log2-scale. Fetal calf serum (FCS) was from PAN-Biotech (Aidenbach, Germany). Unless stated otherwise, all chemicals were from Sigma-Aldrich (St. Louis, MI, USA). The immortalized epithelial endometriotic human cell line 12Z was kindly provided by Prof. Anna Starzinski-Powitz, Frankfurt, Germany [ 34 ]. Primary endometriotic stroma cells were obtained from biopsies of six women with endometriosis who underwent surgical treatment at the Department of Gynecology of Münster University Hospital. Patient characteristics are shown in Table 1 . Primary cells were isolated as described previously [ 35 ]. Cells were cultured in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin in a humidified atmosphere of 7.5% CO 2 at 37 °C. The use of 7.5% CO 2 is consistent with the bicarbonate buffering system of the medium and ensures maintenance of physiological pH during cell culture. A total of 200,000 cells/well of a six-well plate were cultured in DMEM for 24 h. siRNA transfection was performed using Dharmafect reagent (Dharmacon™, Lafayette, LA, USA) according to the supplier’s protocols. This reagent contained 840 µL Opti-MEM™ (Thermo Fisher Scientific, Waltham, MA, USA), 80 µL 20 nM SETD3 siRNA/Opti-MEM™ (Silencer™ Select Pre-Designed siRNA (ID s38639), Thermo Fisher Scientific, Waltham, MA, USA) or negative control siRNA/Opti-MEM™ (Silencer™ Select Negative Control No. 1 siRNA (Cat. no. 4390844), Thermo Fisher Scientific, Waltham, MA, USA), and 80 µL 2.5% Dharmafect/Opti-MEM™ solution in a total volume of 1 mL. Cells were incubated for 24 h at 37 °C and 7.5% CO 2 . The medium was then changed to DMEM with FCS. mRNA and protein extraction were performed 48 h after transfection. mRNA isolation was performed with an InnuPREP RNA mini kit (Analytikjena, Jena, Germany). The mRNA concentration and purity were assessed using an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany). mRNA was then transcribed into cDNA using the cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the supplier’s instructions. For cDNA synthesis 1000 ng of RNA was used for 12Z cells, and 500 ng of RNA was used for primary cells. Quantitative real-time PCR was performed in duplicates for each target gene using Takyon™ ROX SYBR ® MasterMix blue dTTP (Eurogentec, Liège, Belgium), and gene expression levels were measured via a 7300 Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA). Transcriptional analysis was performed using the comparative cycle threshold method and RPS18 was used as a housekeeping gene. Primer sequences are listed in Table 2 . Cell lysates were prepared with a mixture of 222 µL Blue Loading Buffer (Cell Signaling, Cambridge, UK), 22.2 µL 30× Reducing Agent (Cell Signaling, Cambridge, UK) and 422 µL H 2 O for a six-well plate. Then, 15 µL of protein per lane was separated on 10% SDS gels and transferred to nitrocellulose membranes (GE Healthcare, Marlborough, MA, USA). After one hour of blocking with 5% skimmed milk powder in 1× TBST buffer, detection was performed with SETD3 rabbit polyclonal primary antibody (1:1000, Sigma-Aldrich, Darmstadt, Germany, cat. no. HPA003639), β-actin H73 methylation rabbit monoclonal primary antibody (1:1000, Abcam, cat. no. ERP29292-70), β-actin rabbit polyclonal primary antibody (1:1000, Cell Signaling Technology, cat. no. 4967) and anti-rabbit secondary antibody (1:10,000, Sigma-Aldrich, cat. no. 401353). Membranes were subjected to a chemiluminescence reaction, and membranes were stripped with 0.2 M glycine buffer (pH 2.5) afterwards. For tubulin detection as loading control, membranes were subjected to the procedure described above using tubulin mouse antibody (1:10,000, Sigma cat. no. T5168) and anti-mouse secondary antibody (1:10,000, Sigma-Aldrich, cat. no. 401253). For GAPDH detection as loading control, GAPDH mouse antibody (1:1000, Gene Tex, Irvine, CA, USA, cat no. GTX627408) and anti-mouse secondary antibody (1:10,000, Sigma-Aldrich, cat. no. 401253) were used. Cell viability was determined after treatment with control or SETD3 siRNA by the MTT (methylthiazolyldiphenyltetrazolium bromide) assay. SETD3 -depleted or control cells (4 000 maximum) were plated in 96-well plates with DMEM medium without phenol red (Gibco ® , Thermo Fisher Scientific, Waltham, MA, USA) with FCS and incubated for 96 h at 37 °C. Then, the medium was changed to 20 µL of MTT solution (250 mg MTT powder, Sigma-Aldrich, cat. no. 5655, in 50 mL PBS) per well. After 4 h of incubation, 100 µL of a stopping solution (50% N,N-Dimethylformamid and 10% SDS in H 2 O) per well was applied. Photometric analysis was performed after a 24 h incubation period at room temperature in a VersaMax ® ELISA Microplate Reader (Molecular Devices, San José, CA, USA) at a wavelength of 595 nm. The MTT assay was performed with 12Z cells and primary cells from four patients. SETD3 knockdown cells and controls were cultivated in DMEM with FCS in six-well plates in duplicates. Adherent cells were washed with PBS and a cross-shaped scratch was performed using a sterile 1000 µL pipette tip. The media was changed to remove detached cells, followed by a 24 h incubation at 37 °C. Cell migration into the free surface area was determined at time intervals of 0, 5, 10, and 24 h after the initial scratch. Images of the surface areas were taken under an Axiovert 100 microscope using the Axio Vision program (Carl Zeiss, Jena, Germany; https://www.micro-shop.zeiss.com/en/us/ , accessed on 1 March 2026). The ratio of cell migration was calculated as the percentage of the free surface cross-area at 24 h compared with the area of the initial scratch at 0 h. ImageJ 1.x software (National Institutes of Health, Bethesda, MD, USA) was used for surface area measurement. The scratch wound assay was performed with 12Z cells and primary cells from four patients. SETD3 -depleted primary cells or controls were diluted to 400,000/300 µL DMEM. Then, collagen gels were prepared on ice using 714 µL collagen type I (4.88 mg/mL, Corning, cat. no. 354236, Corning, NY, USA), 16.4 µL NaOH, 169 µL H 2 O, and 100 µL 10X PBS for each SETD3-depleted cells and controls. A total of 250 µL of the respective cell suspension was mixed with the collagen gel to create cell-embedded collagen gels with a cell–collagen ratio of 1:4. The gels were transferred into a 24-well plate (500 µL/well) and incubated at 37 °C for 1 h to allow polymerization. Then, 1 mL of DMEM with FCS was added to each well and incubated at 37 °C. Photographs were taken at 24 h intervals using a digital camera (resolution of 3024 × 4032 pixels) and an Axiovert 100 microscope (Carl Zeiss, Jena, Germany) to monitor the contraction progress. The collagen surface areas of the gels were measured using the ImageJ 1.x program (National Institutes of Health, Bethesda, MD, USA). As the assay required high cell numbers, it was performed with stroma cells of a single patient (OP23) only. SETD3 -depleted 12Z cells and controls were diluted to 100,000 cells/2 mL in DMEM with FCS. Then, 500 µL (25,000 cells) were transferred to Matrigel chambers (Corning ® BioCoat™ Matrigel ® Invasion Chambers, cat. no. 354480, Corning, NY, USA) and incubated for 24 h at 37 °C. Subsequently, the media was carefully replaced by FCS-free DMEM and invasion was initiated by filling the Matrigel wells with 750 µL of DMEM with FCS. After another 24 h incubation period, the media were removed, and cells were fixed in methanol and washed with PBS. The cells were then stained in 1% toluidine blue in SB buffer (Sigma, cat. No. T3260) for six minutes and washed with H 2 O. For each membrane, cells in two visual fields were counted under an Axiovert 100 microscope (Zeiss, Jena, Germany; magnification 10×). The Matrigel invasion assay was performed with 12Z cells. SETD3 -depleted 12Z cells and controls were placed in a 96-well plate. Seeding densities were optimized for each cell type to achieve comparable confluency and imaging quality at the time of analysis (10,000 cells each for 12Z cells and 7500 cells each for primary cells from one patient). All media were removed, and cells were fixed in a 4% formaldehyde solution (100 µL/well) for 10 min, then washed with 1 mL PBS for 5 min and incubated in 0.1% Triton (500 µL/well; Sigma-Aldrich, USA) for 10 min. Washing was repeated, followed by a 25 min incubation in 1% BSA/PBS (Thermo Fisher Scientific, Waltham, MA, USA). Actin filaments were stained with Phalloidin CruzFluor™ (1:1000, Santa Cruz Biotechnology, Dallas, NY, USA) in blocking solution (1% BSA, 10% FCS, 0.02% Triton) for 60 min. Washing was repeated before cells were stained with DAPI (1 µL in 25 mL PBS) for 10 min. Washing was repeated. Cells were then conserved in 100 µL PBS/well and examined by immunofluorescence microscopy on a Keyence BZ-X1000 Series Microscope (Keyence Corporation, Osaka, Japan). For immunofluorescence quantification, the integrated fluorescence intensity was measured and normalized to the number of DAPI-positive nuclei in the corresponding field of view. All procedures were performed following national and international guidelines and approved by the federal state of Mecklenburg Western-Pommerania, Germany (approval no. AZ 7721.3-2-015/20). Female mice, bred at the Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany, were housed in groups of three animals per cage. The FL1 line, a mouse line selected for high fertility, was compared to non-selected control line, as described in references [ 27 , 36 , 37 , 38 ]. A commercial breeding diet for rodents and water was provided ad libitum, and illumination of animal facilities was between 6.00 a.m. and 6.00 p.m. A male mouse was kept for acoustic, visual and olfactory stimulus in a separate cage. To avoid cycle-related alterations of gene expression, samples were taken exclusively in estrus. Vaginal cytology was used to determine the stage of the estrous cycle like previously described [ 27 , 36 , 37 ]. Mice at the age of 9–13 weeks were euthanized by nitrogen inhalation. After determination of death, samples were taken. The uterus was cleaned from fat tissue and immediately snap-frozen and stored at −70 °C. Samples were pulverized in liquid nitrogen. RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and RNA quality was assessed using the A260/A280 ratio. Only samples with ratios between 2.0 and 2.1 were included for further analyses. cDNA was synthesized from 400 ng of total RNA using an iScript cDNA Synthesis Kit (Biorad, München, Germany) according to the manufacturers protocol. Primers were designed using Primer-BLAST ( https://tib-molbiol.com/ , accessed on 1 March 2026) and purchased from TIB Molbiol (Berlin, Germany). Samples were analyzed in duplicates using 4 µL primer mix, 5 µL SsoAdvancedTM Universal SYBR Green Supermix (Biorad, München, Germany) and 1 µL of cDNA reaction solution and was loaded onto a 96-well plate and amplified by real-time PCR (iCycler, Biorad, München, Germany). RT-qPCR was performed using the following cycling conditions: initial denaturation at 94 °C, followed by 40 cycles of 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 45 s. Amplification specificity was verified by melt curve analysis. Only reactions exhibiting a single distinct melting peak were considered specific and included in the analysis. The results of the mRNA abundance are calculated relative to a combination of the reference genes 36B4 , RPS18 and B2m . Relative gene expression and statistical analysis was calculated using the Relative expression software tool, version REST 2009 [ 38 , 39 ]. Control samples served as calibrators and were therefore set to 1. Primer sequences are listed in Table 3 . The data were tested for significance employing Student’s unpaired t -test. RT-qPCR data in the mouse model were analyzed using REST (Relative Expression Software Tool), version REST 2009 [ 38 , 39 ]. All experimental assays were repeated at least three times in duplicates. Data are presented as the mean values ± SEM or SD (animal model). Significant p -values are indicated as follows: p < 0.05 by one asterisk *, p < 0.01 by two asterisks ** and p < 0.001 by three asterisks ***. This study was approved by the local ethics committee (Ethikkommission der Ärztekammer Westfalen-Lippe und der Medizinischen Fakultät der Westfälischen Wilhelms-Universität Münster; reference no. 1 IX Greb from 19 September 2001 and updated on 6 December 2012) and reference number 2025-693-f-S (2025). No generative artificial intelligence (GenAI) has been used in this paper.

Endometriosis is a benign but invasive disease that affects women of reproductive age, resulting in a wide range of symptoms including severe pain and subfertility [ 1 , 2 , 3 ]. It has been demonstrated that endometriotic cells share cell biological characteristics with malignant tumors [ 4 ], in which case tumor cells are characterized by increased motility and migration, enabled by alterations of the cytoskeleton [ 5 ]. Hence, the cytoskeleton is likely involved in the pathogenesis of endometriosis, particularly deep endometriosis, promoting invasive cell behavior and the formation of ectopic lesions [ 6 ]. Indeed, previous work from our laboratory has shown that microRNA-dependent regulation of the actin bundling protein fascin and Neural Wiskott–Aldrich Syndrome protein N-WASP/WASL , a regulator of actin filament formation, regulates invasive growth of endometriotic cells [ 6 ]. Actin is the major protein of the cytoskeleton, essential for cell growth, division and migration [ 5 , 7 ]. Regulation of actin is extremely complex but indispensable for cytoskeletal integrity, and methylation is among the various mechanisms of actin regulation [ 7 , 8 ]. Post-translational methylation is one of the most common modifications in eukaryotic protein synthesis. Methylation of histones on arginine and lysine residues is well known, allowing for epigenetic regulation of gene expression [ 7 ]. However, methylation can also occur on non-histone proteins, which enables cell regulation apart from the DNA level. Methylation of β-actin was discovered in 1967 [ 9 , 10 ], but the enzyme responsible was only recently identified. The ubiquitously expressed protein SETD3 (SET domain containing 3) has been reported as actin-specific histidine N-methyltransferase [ 7 , 11 ] SETD3 catalyzes methylation of β -actin at His73 with the use of S-adenosyl methionine as methyl donor, leading to the stabilization of actin filaments [ 8 , 11 ]. Interestingly, SETD3 knockdown animal models are still viable, and SETD3 therefore does not impair the basal functions of actin [ 11 , 12 ]. Moreover, SETD3 is described to have various protein interactions whose underlying pathways are still unknown. At least 172 SETD3 interacting proteins have been identified but remained largely unstudied [ 13 ]. It is reported that SETD3 is involved in the positive regulation of p53 [ 14 ], as well as in the upregulation of VEGF (Vascular endothelial growth factor) through methylation of FoxM1 (Forkhead box protein M1) [ 13 ], the latter driving tumor proliferation and metastasis [ 15 , 16 ]. These properties indicate a potential role of SETD3 in angiogenesis and oncogenesis, in accordance with altered SETD3 expression levels in different carcinomas [ 17 , 18 , 19 ]. Liver cancer proliferation was enhanced by elevated SETD3 levels as a result of inhibited degradation as SETD3 itself is normally subject to a cell cycle-dependent downregulation by FBXW7 (F-box/WD repeat-containing protein 7) and GSK-3 (glycogen synthase-kinase 3) [ 20 ]. Due to these properties, we hypothesize that alterations in SETD3 expression may affect cytoskeletal function and cell motility not only in malignant diseases but also in benign invasive diseases such as endometriosis. Furthermore, we state that our work is the first study on SETD3 and endometriosis.

No studies so far have investigated the relevance of SETD3 for endometriosis. Prior to analyzing SETD3 function in vitro, we analyzed the mRNA expression of SETD3 in 115 endometriosis patient tissues and 53 control tissues via EndometDB [ 21 ]. Analysis compared the expression in healthy control tissue to endometriotic tissue from differently localized lesions. Although data did not show any significant difference, the highest SETD3 expression was found in deep infiltrating endometriosis tissue ( Figure 1 ). This finding prompted us to investigate a possible functional role for SETD3 in endometriotic cells. We employed SETD3 siRNA knockdown in vitro in the immortalized endometriotic cell line 12Z as well as in primary endometriotic stroma cells. Successful SETD3 knockdown was confirmed by Western blot ( Figure 2 A,B) and RT-qPCR ( Figure 2 C,D). Following the downregulation of SETD3 , no significant differences in the cell viability of 12Z and primary cells were noted ( Figure 3 A). However, we found significant restrictions in all other experiments with relevance for invasive cell behavior. The ability to contract collagen was significantly reduced in SETD3 -depleted primary cells compared to control after 72 h (54.17% vs. 32.15%) and 96 h (41.03% vs. 24.16%) ( Figure 3 B). These findings were consistent with data obtained by the scratch wound assay, where both types of SETD3 knockdown cells showed a significant delay in wound closure after 24 h compared to the control (59.4% vs. 46.5% for 12Z cells and 87.3% vs. 78% for primary cells) ( Figure 3 C). The Matrigel invasion assay revealed a significant reduction in invasiveness of SETD3 siRNA treated 12Z cells by 72% with respect to control ( Figure 3 D). Next, we performed fluorescence microscopy using phalloidin and DAPI staining ( Figure 4 ). SETD3 knockdown was associated with a reduction in phalloidin fluorescence intensity per cell compared with control cells; however, this difference did not reach statistical significance (7.121 ± 0.3437 vs. 6.015 ± 0.392, p = 0.1011 and 4.619 ± 0.2892 vs. 2.970 ± 0.6748, p = 0.0880). Actin stress fibers seemed to be less frequent in siRNA knockdown in primary cells. These findings support a possible role for SETD3 in maintaining filamentous actin cytoskeletal architecture in endometriotic cells. Consistent with these findings, Western blot analysis revealed a significant reduction in β-actin histidine-73 methylation ( Figure 5 A) following SETD3 silencing in both 12Z cells (relative protein expression: 0.294 ± 0.069 vs. 0.9997 ± 0.115, p = 0.0079) and primary stromal cells (relative protein expression: 0.727 ± 0.035 vs. 1.0 ± 0.06, p = 0.013), whereas total β-actin protein levels remained unchanged ( Figure 5 B). Together, these results indicate that SETD3 contributes to the maintenance of F-actin organization in endometriotic cells, likely through regulation of β-actin methylation rather than changes in total β-actin expression. Fascin , ACTB (beta-actin), ACTG (gamma-actin), ASMA (alpha smooth muscle actin), FOXM1 (Forkhead-Box protein M1), FBXW7 (F-box/WD repeat-containing protein 7) and GSK-3 (glycogen synthase kinase 3) are proteins with relevance for invasive diseases, some of them regulated by or regulating SETD3 [ 11 , 13 , 20 , 21 , 22 , 23 ]. We performed RT-qPCR. Gene expression analysis revealed no significant alterations in 12Z cells or primary cells ( Figure 6 ). Apart from pain, subfertility is associated with endometriosis [ 2 , 3 ]. To investigate a possible association of SETD3 with fertility, we analyzed its expression in the uteri of a unique mouse model associated with superfertility, the Dummerstorf superfertile mouse line FL1. This mouse line 1 was selected for increased litter size and the total birth weight of the litters for almost 200 generations [ 24 , 25 ]. During the selection process FL1 mice almost doubled the number of pups per litter, as well as the total birth weight of the entire litters compared to the unselected control line (ctrl) in the first parturition and show no signs of growth retardation in the offspring [ 26 ]. FL1 females ovulate approximately twice as many oocytes compared to ctrl mice [ 27 ]. Furthermore, not only do FL1 mice deliver a high number of pups in the first pregnancy, they are able to deliver large litter sizes with a high reproductive mating rate over a long-time period without health issues [ 28 ] and therefore are a worldwide unique animal model for increased female reproductive performance, high fertility and longevity. To analyze SETD3 expression, uteri of 10 FL1 and 9 control mice were collected during estrus and snap-frozen, followed by RNA isolation, reverse transcription and qPCR analysis for SETD3 expression. A significant, 1.6-fold downregulation of SETD3 was observed in the uteri of superfertile mice compared to controls ( Figure 7 ), indicating a possible connection of SETD3 expression to fertility in female mice.

SETD3 has been identified to be an actin-specific histidine N-methyltransferase [ 7 , 11 ]. Structural analysis has indicated that methylation regulates actin’s flexibility and stability [ 8 ], and the relevance of SETD3 has been studied for various cancer types [ 29 , 30 ]. These properties make SETD3 a promising candidate to participate in the development of endometriosis. Altered expression levels of SETD3 have been found in different cancer cells; it is reported that aberrant SETD3 levels promote tumor proliferation in hepatocellular carcinoma, in line with expressional changes of SETD3 described for renal, ovarian and breast cancer in relation to altered patient survival [ 17 , 18 , 19 , 30 ]. Gene expression analysis of SETD3 is useful to evaluating its clinical potential as biomarker and to obtaining initial data on its relevance for endometriosis. Although SETD3 expression was not significantly different between endometriosis subtypes in this study, a trend towards higher expression in deep-infiltrating lesions was observed. Interestingly, our functional experiments demonstrated that SETD3 contributes to migration, invasion, and collagen contraction, processes that are characteristic of the invasive phenotype of deep endometriosis. As a specific substrate of SETD3 , β-actin is methylated and thus stabilized under physiological conditions [ 7 ]. Following the downregulation of SETD3 , we assumed that the stabilization of actin filaments was no longer applicable, which may have led to a decrease in cytoskeletal stability. We were able to demonstrate a significant reduction in histidine-73 methylated β-actin following SETD3 knockdown in both 12Z cells and primary endometriotic stromal cells, whereas total β-actin protein levels remained unchanged. These findings confirm that SETD3 depletion impairs β-actin methylation rather than overall β-actin expression and provide mechanistic evidence linking SETD3 activity to cytoskeletal regulation in endometriotic cells. Expression of unmethylated actin isoforms, ACTB , ACTG and ASMA , was not altered in our study while SETD3 is described to potentially methylate all actin isoforms in vitro [ 11 ]. Strong results for the effect of SETD3 depletion on cytoskeletal function were observed in our functional experiments. Endometriotic cells lacking SETD3 showed decreased cell motility, collagen contractility and invasiveness. In agreement with that, the knockdown of SETD3 in breast cancer cells markedly reduced invasiveness and collagen contraction ability [ 30 ]. These findings indicate that reduced SETD3 expression could affect cytoskeletal function not only in malignant but also in benign invasive diseases, more precisely in endometriosis. Cell viability was not significantly decreased in our cells, but SETD3 deprivation is known to reduce cell proliferation in breast cancer and liver cancer cells [ 20 , 30 ]. Other studies revealed reduced cell viability in accordance with SETD3 overexpression, characterizing SETD3 as an apoptotic regulator [ 31 ]. SETD3 initiates apoptosis by the positive regulation of p53, which characterizes SETD3 as a bivalent regulator of cell viability depending on the particular entity [ 14 ]. For endometriosis, various studies reported a reduction in apoptosis during menstruation which facilitates the formation of ectopic lesions [ 32 , 33 ]. Therefore, potential apoptotic effects of SETD3 in endometriosis are worth addressing in future studies. Interestingly, most of SETD3 protein is localized in the cytoplasm of the cell [ 12 , 20 ]. To further investigate the consequences of impaired β-actin methylation on cytoskeletal organization, we performed phalloidin staining to visualize filamentous actin. SETD3 knockdown resulted in reduced phalloidin fluorescence intensity in both 12Z cells and primary endometriotic stromal cells. Importantly, these alterations occurred despite unchanged total β-actin protein levels, suggesting that SETD3 primarily affects the functional state and organization of filamentous actin rather than its overall abundance. Together with the observed reduction in β-actin H73 methylation, these findings support a model in which SETD3 -dependent actin methylation contributes to the maintenance of F-actin architecture and cytoskeletal dynamics required for cell migration, invasion, and collagen contraction. It is known that SETD3 is not necessary for basal actin function as SETD3 knockdown animals are still viable [ 11 , 12 ]. Downregulation of SETD3 may therefore not affect basal actin morphology in endometriotic cells but may result in reduced actin methylation potentially modifying the functional status of actin and, subsequently, in reduced cell motility, contractility and invasiveness. To assess a potential link between SETD3 and fertility, we employed a unique and informative resource, the Dummerstorf superfertile mouse model FL1 [ 24 , 25 , 26 , 27 ]. In line with our in vitro and clinicopathological investigation, SETD3 expression was significantly downregulated in FL1 mice compared to normal controls. If applied to the situation in humans, in an endometriosis context, a low expression of SETD3 in uterine tissue (specifically the endometrium) could reduce the occurrence of endometriosis by hampering invasive growth and therefore establishment of the endometriotic lesion when distributed via classical etiological routes such as retrograde menstruation. A reduction in endometriotic lesions would in turn reduce a number of pathogenetic mechanisms thought to be involved in endometriosis-associated infertility, including an enhanced local inflammatory response, which may indirectly affect the ovarian reserve, and anatomical distortions due to adhesions and fibrosis [ 1 , 2 ]. Interestingly, although the expression of SETD3 in the uterus of FL1 mice is decreased, it has been shown that SETD3 mRNA expression is significantly increased in ovaries [ 26 ] and in granulosa cells of antral follicles (Ludwig CLM et al. unpublished) of FL1 mice. Thus, while decreased mRNA levels of SETD3 in the uterus potentially prevent invasive growth of endometriotic tissue, increased SETD3 levels might inhibit apoptotic pathways in the ovary and in granulosa cells, which in turn leads to increased follicular survival, improved follicular development and therefore higher ovulation rates in FL1 mice. However, it should be taken into account that the high SETD3 /low apoptotic ovarian phenotype of FL1 mice is not associated with any negative health issues. Finally, SETD3 may also affect fertility at very late stages of pregnancy as SETD3 -deficient female mice have severely decreased litter sizes due to primary maternal dystocia [ 11 ]. However, we consider a complete knockout of SETD3 in humans a rare event, whereas the more moderate regulation observed in the Dummerstorf superfertile FL1 mouse appears to be closer to a physiological regulation. Our study demonstrated that SETD3 depletion reduces β-actin histidine methylation and is accompanied by impaired F-actin organization, decreased migration, invasion, and collagen contraction. Together, these findings identify SETD3 -mediated β-actin methylation as an important regulator of cytoskeletal dynamics in endometriotic cells and suggest that SETD3 may contribute to the invasive phenotype characteristic of endometriosis.