Cell consequences of loss of function of the epigenetic factor EHMT1

Cellular Signalling 108 (2023) 110734 Available online 29 May 2023 0898-6568/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Cell consequences of loss of function of the epigenetic factor EHMT1 Lucía Iglesias-Ortega a , 1 , Clara Megías-Fern´ andez b , c , 1 , Paloma Domínguez-Gim´ enez c , Silvia Jimeno-Gonz´ alez b , c , Sabrina Rivero a , c , * a Departamento de Citología e Histología Normal y Patol´ ogica, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain b Departamento de Gen´ etica, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain c Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Junta de Andalucía-Universidad Pablo de Olavide, Sevilla, Spain ARTICLE INFO Keywords: Epigenetics Neurodevelopment Kleefstra syndrome EHMT1 Cell biology ABSTRACT EHMT1 is an epigenetic factor with histone methyltransferase activity that appears mutated in Kleefstra syndrome, a neurodevelopmental genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features. Despite recent progress in the study of the function of this gene and the molecular etiology of the disease, our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited. Here, we show that EHMT1 depletion in RPE1 cells leads to alterations in the morphology and distribution of different subcellular structures, such as the Golgi apparatus, the lysosomes and different cell adhesion components. EHMT1 downregulation also increases centriolar satellites detection, which may indicate a role for EHMT1 in centrosome functioning. Furthermore, the migration process is also altered in EHMT1 depleted cells, which show reduced migration capacity. We consider that the described phenotypes could open new possibilities for understanding the functional impact of EHMT1 haploinsufficiency in Kleefstra syndrome, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in this neurodevelopmental disorder. This knowledge could be relevant not only for the treatment of this syndrome, but also for other neurodevelopmental conditions that could share similar deregulated cellular pathways. 1. Introduction During embryonic development, cells differentiate, acquiring a characteristic gene expression pattern that defines their cellular identity. This process is mediated, at least in part, by different epigenetic mechanisms that ultimately determine how our genes are expressed. Now we also know that epigenetic mechanisms determine cell plasticity [1]. For example, the expression of genes associated with the neuronal plasticity necessary for cognitive processes and memory consolidation, both during development and throughout adult life, is regulated by chromatin remodeling [2]. Consequently, more than 50 chromatin factors have already been identified whose alteration is associated with different syndromes that arise with intellectual disability and behaviors of the autism spectrum disorder [3–5]. One of those proteins is EHMT1 (also known as GLP, for G9a-like protein, or KMT1D), which is mutated in Kleefstra syndrome (OMIM #610253) (also known as 9q34.3 deletion syndrome), a rare genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features [6]. Kleefstra syndrome has been described to be caused by haploinsufficiency of the EHMT1 gene, which encodes a histone methyltransferase that, together with its paralog EHMT2 (also known as G9a or KMT1C), catalyzes histone 3 lysine 9 (H3K9) mono- and di-methylation [7], a modification associated with transcriptional repression [8]. EHMT1 appears to be a key regulator of memory consolidation. The loss of EHMT1 function in mice and Drosophila has been reported to cause learning and memory impairments [9,10]. In mice, Ehmt1 haploinsufficiency, which recapitulate autistic-like features that are seen in patients with Kleefstra syndrome, leads to synaptic dysfunction associated with a significant reduction in dendritic arborization and the number of mature spines in specific hippocampal neurons [10]. These structural abnormalities, which constitute prominent features in patients with intellectual disability, are known to be associated with impaired cytoskeleton remodeling, since the dynamic morphology of actin-rich dendritic spines is the consequence of continuous * Corresponding author at: Departamento de Citología e Histología Normal y Patol´ ogica, Facultad de Medicina, Universidad de Sevilla, Avenida S´ anchez-Pizju´ an s/ n., 41009 Sevilla, Spain. E-mail address: [email protected] (S. Rivero). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig https://doi.org/10.1016/j.cellsig.2023.110734 Received 16 September 2022; Received in revised form 20 May 2023; Accepted 23 May 2023 Cellular Signalling 108 (2023) 110734 2 polymerization and breakdown of actin filaments and dynamic microtubules in spine [11]. Furthermore, a reduced ability to establish synaptic scaling, a specific form of homeostatic plasticity, has been detected [12]. In this context, EHMT1 has been shown to repress the expression of Bdnf (brain-derived neurotrophic factor), an immediate early gene implicated in long-term memory formation, by increasing H3K9me2 at the Bdnf promoter [12]. Ehmt1 haploinsufficiency in mice also leads to alterations in neuronal network activity dependent on the N-methyl-D aspartate (NMDA) receptor, which constitutes with the 2-amino-3-hy- droxy-5methyl-4-isoxazole propionic-acid (AMPA) receptor, the two main types of glutamate receptors found in the postsynaptic dendritic spines of excitatory synapses. Additionally, it has been described that Ehmt1 haploinsufficiency activate in mice neuroinflammation induced by the Caspase-1-IL-1β pathway [13]. This study also showed that postnatal neuron specific EHMT1 supply reversed some of the neurological phenotypes presented in Ehmt1 Δ/+ mice, although this was not sufficient to improve abnormal behaviors observed in these mice, suggesting that they are caused by insufficient expression of EHMT1 in both neuronal and non-neuronal cells [13]. Mutation of this gene in Drosophila leads to reduced dendrite branching and memory deficits associated with reduced methylation of genes with a known role in these processes [9]. Improvement of the neurological defects was also achieved by conducting a gene rescue at the adult stage in a Drosophila model of Kleefstra syndrome [9]. In the HEK293T cell line, CRISPR generated EHMT1 haploinsufficiency to assess a known pathogenic variant seen in patients, leads to transcriptional deregulation of different genes related to neuronal development and other cellular pathways such as DNA repair, unfolded protein response (UPR), Wnt/β-catenin signaling and protein secretion [14]. Neurons induced from iPS cells of patients with Kleefstra syndrome also showed altered neural network activity and transcriptional deregulation of neuronal developmental genes [15]. In this context, reduced EHMT1 activity has been shown to decrease NRSF/REST protein, a neuron-specific gene regulator, via upregulation of microRNA (miRNA)-mediated suppression of NRSF/ REST protein synthesis [16]. Mutations in EHMT1 have also been associated with aberrant expression of MML3, SMARCB1, NR1I3, and MBD5, which encode for epigenetic regulators that interact with EHMT1 [17]. However, despite the progress made in recent years in the study of the function of this gene [12,18–23], our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited, so the design of a causal treatment remains a challenge. In this study, to understand the link between epigenetic regulation of EHMT1 and the underlying cellular mechanisms that lead to Kleefstra syndrome, we have studied the effects of reduced EHMT1 activity in RPE1 cells on various subcellular structures known to be affected in other intellectual disability disorders, such as different membrane organelles and cytoskeleton elements. We have also examined the effect of EHMT1 reduction on cell migration, a key process in neurogenesis. With these approaches, we have identified cellular alterations that might help to understand the underlying consequences of EHMT1 haploinsufficiency, opening new lines of research for the treatment of patients affected by this disease. 2. Materials and methods 2.1. Cell lines and growth conditions RPE1 cells, human epithelial cells from non-tumor retinal pigment epithelium (kindly provided by Prof. Pablo Huertas), were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). HeLa cells (kindly provided by Prof. Rosa Ríos) were cultured in DMEM medium supplemented with 10% fetal bovine serum. Cells were maintained at 37 ◦C and 5% CO 2 and mycoplasma infection was tested using the MycoAlert detection kit (Lonza). Cells were routinely treated with anti-mycoplasma antibiotic Plasmocin (InvivoGen) before freezing. 2.2. Cell transfection For transient transfection, lipofectamine RNAiMAX (Invitrogen) was used following the protocol recommended by the manufacturer. siRNAs were used at a final concentration of 30 nM (Control 5´-CGUACGCG- GAAUACUUCG-3′; EHMT1 5′-GGACGGUGAUUGAGAUGUUTT-3′). All assays were performed 72 h after transfection. 2.3. Cell viability assay and cell cycle analysis Cells were seeded in 6-well plates starting at 8 ×10 4 cells per well and incubated for 24 h. After siRNA transfection for 72 h, cells were counted using an automatic cell counter (Bio-Rad). Cell cycle was determined by flow cytometry as previously described [24] using a FACSCalibur (BD, Franklin Lakes, NJ, USA). Cell cycle distribution data were further analyzed using MODFIT LT 3.0 software (Verity Software House Inc., Topsham, ME, USA). 2.4. RNA extraction and RT-qPCR For RNA extraction, the RNeasy Kit (Qiagen) was used, following the manufacturer’s instructions. DNase I digestion (Promega) was included to avoid possible DNA contamination. cDNA was generated from 500 ng of total RNA using Maxima H minus (Thermo Fisher Scientific). Realtime quantitative PCR (RT-qPCR) was performed with the Applied Byosistems 7500 FAST real-time PCR system, using the Applied Byosistems Power SYBR Green Master Mix. Values were normalized to the expression of the GAPDH housekeeping gene. Primer sequences are the following: EHMT1 up 5´-GAGATCACCACACTGGCCAA-3′; EHMT1 low 5´- GTCTTCACACAGCACGAGGA-3′. GAPDH up 5´-GAGTCAACG- GATTTGGTCGT-3′; GAPDH low 5´-AATGAAGGGGTCATTGATGG-3′. 2.5. Western blot Western blot experiments were performed as previously described [25,26] with antibodies anti-EHMT1 (A301642A1, Bethyl Laboratories), anti-GRASP65 (sc-374432, Santa Cruz), anti-Vinculin (sc-73614, Santa Cruz), anti-β-Catenin (sc-7936, Santa Cruz), anti-Vimentin (sc-6260, Santa Cruz), anti-LAMP-1 (sc-20011, Santa Cruz) and antiα -Tubulin (T9026, Sigma). 2.6. Immunofluorescence and image analysis For immunofluorescence experiments, cells were cultured on glass coverslips and fixed in a 4% paraformaldehyde solution in PBS (Santa Cruz) following a previously described protocol [25]. Primary antibodies used were anti-EHMT1 (A301642A1, Bethyl Laboratories and AB1969, Abcam), anti-GRASP65 (sc-374432, Santa Cruz), anti-Vinculin (sc-73614, Santa Cruz), anti-β-Catenin (sc-7936, Santa Cruz), anti- Vimentin (sc-6260, Santa Cruz), anti-LAMP-1 (sc-20011, Santa Cruz) and antiα -Tubulin (sc-23948, Santa Cruz). Secondary antibodies used were Alexa Fluor 488 anti-mouse IgG (A-11029, Invitrogen), Alexa Fluor 488 anti-rabbit IgG (A-11034, Invitrogen), and Alexa Fluor 568 antirabbit IgG (A-11011, Invitrogen). Cells were examined under a motorized upright wide-field microscope (LEICA DM6000B). Confocal images were acquired in a TCS SP5 Leica confocal with an HCX PL APO Lambda blue 63×/1.4 OIL objective (at Zoom 1 for quantification or at Zoom 3 for Figures). Image processing was carried out using the Leica (LAS) and Adobe Photoshop software. Images were analyzed with Fiji (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imageg.nih.goc/ij/, 1997–2018). In order to analyze the changes observed in the Golgi apparatus of EHMT1 siRNA transfected cells, with a collapse/shrunken shape compared to the elongated curved shape in control cells, the circularity measurement function in ImageJ L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 3 was used, considering a minimum area of 35 pixels. A value of 1.0 indicates a perfect circle. The analyze particles tool in Fiji, setting a particle size of 0–5 pixels, were used to analyze intensity values of the LAMP-1 vesicles. 2.7. Migration assays Migration assays were performed as previously described [24]. Twowell inserts (Ibidi) were used in each of which 70 μ l of a cell preparation was added at a concentration of 300,000 cells/ml. Twenty-four hours later, the inserts were removed, and the resulting wound closure was monitored. Images were acquired using an inverted microscope with a 10×objective (Leica DMi6 Hamamatsu ORCA-ER camera). 3. Results and discussion 3.1. Cell consequences of chromatin modifier EHMT1 reduction EHMT1 is a ubiquitously expressed protein with lysine methyltransferase activity that regulates histone methylation and euchromatin formation [27]. Recent studies have allowed the identification of many genes whose expression is regulated by EHMT1. In fact, it has been Fig. 1. Efficiency of EHMT1 depletion in RPE1 cells through specific siRNAs. (a) Relative expression of EHMT1 mRNA levels quantified by RT-qPCR. To facilitate the comparison, data were normalized to control cells. (b) Immunofluorescence staining of EHMT1 in RPE1 cells transfected with the indicated siRNAs. Bars, 25 μ m. (c) Depletion was measured 72 h after siRNA transfection by western blot with the indicated antibodies. α -Tubulin was used as a loading control. (d) Images of RPE1 cell cultures before (0 h) and 72 h (72 h) after siRNAs transfection. (e) Growth curve of RPE1 cells transfected with the indicated siRNAs. (f) Cell cycle distribution after EHMT1 downregulation. The resultant percentage of cells were plotted. Values are the average and SEM from three independent experiments. Statistical significance was calculated using a t-test (a, e) or Two-way ANOVA test (f) (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ns, not significant). L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 4 recently identified the existence of share gene expression disruptions resulting from mutations in genes that encode different chromatinmodifying proteins, including EHMT1, CHD8, CREBBP, or NSD1, all of which are implicated in the genesis of different neurodevelopmental disorders [28]. However, little is known about the underlying cellular mechanisms that result in these disorders. To help define the underlying cellular mechanisms that lead to Kleefstra syndrome, we first investigated whether downregulation of the Kleefstra syndrome-associated protein EHMT1 results in changes in different cell structures and organelles whose defective function has been recognized in different intellectual disability disorders, such as the Golgi apparatus, the lysosomal system, the cytoskeleton, or the centrosome [29]. We performed immunofluorescence imaging to study the morphology and localization of these structures using different known markers in cells in which EHMT1 expression was reduced with specific siRNAs. We used the well characterized RPE1 cell line, an immortalized non-tumor human epithelial cell line commonly used in cell biology studies that has the advantages that can be routinely cultured in the laboratory and is suitable for siRNA transfection. The reduced expression after transfection of EHMT1 siRNAs is evident at both the RNA level and the protein level (Fig. 1A-C). Surprisingly, a marked effect of EHMT1 reduction on cell proliferation was observed 72 h after transfection (Fig. 1D). Cell growth assays and cell cycle distribution analysis showed that under EHMT1 knockdown RPE1 cells showed significant inhibition of growth and S-phase cell cycle arrest compared with control cells (Fig. 1E, F), which may indicate that EHMT1 is involved in cell cycle regulation in this cell line. 3.1.1. EHMT1 is necessary for the maintenance of the Golgi apparatus structure Given the fundamental role of the Golgi apparatus in vital cell processes such as protein processing and trafficking, migration, and ciliogenesis, and the importance of its structure for the normal development of such functions [30], its study may provide clues to discover the involvement of EHMT1 in these cell processes. Therefore, to study the effect of a reduction on EHMT1 expression in the morphology and distribution of the Golgi apparatus, an antibody that recognizes GRASP65, a peripheral protein on the cis face of the Golgi, was used. Interestingly, although the organelle maintains its perinuclear location, the characteristic crescent-shaped structure observed in RPE1 was lost under EHMT1 depletion, and most cells showed a morphological change with a collapsed Golgi around the nucleus (Fig. 2). To explore whether the Fig. 2. EHMT1 is necessary for the maintenance of the Golgi apparatus structure. (a, b) RPE1 cells transfected with the indicated siRNAs were labelled for the cis Golgi marker GRASP65 (green) and EHMT1 (red). Image overlay with DAPI-stained nuclei is shown in Merge. Bars, 25 μ m or 7.5 μ m in images of higher magnification. (b) Quantification of immunofluorescence experiments shown in a. Golgi morphology was analyzed using circularity measurement function in ImageJ in a total number of cells per condition ≥150. A value of 1.0 indicates a perfect circle. (c) Western blot analysis of EHMT1, GRASP65 and α -tubulin in RPE1 cells after transfection with the indicated siRNAs. (d) Quantification of EHMT1 and GRASP65 levels, normalized to GAPDH expression. Values are the average and SEM from three independent experiments. Statistical significance was calculated using a t-test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ns, not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 5 observed alterations are exclusive to RPE1 cells, we transfected EHMT1 siRNAs into HeLa cells, a tumorigenic cell line in which the Golgi usually appears as a compact perinuclear structure. In this case, after EHMT1 reduction, there was a clear disruption of the Golgi that appears fragmented and dispersed throughout the cell cytoplasm (Supplementary Fig. S1). Together, these findings may indicate that EHMT1 activity plays a crucial role in the maintenance and function of the Golgi in these cell lines. 3.1.2. EHMT1 depletion leads to lysosomes accumulation Lysosomes are key organelles for the maintenance of cellular homeostasis in which essential metabolic and cell signaling functions are carried out. Lysosomes are known to have the ability to respond to intra- and extracellular stimuli that can result in changes in their number, size, and intracellular position [31]. To study the effect of the decrease in EHMT1 on the morphology and distribution of the lysosomal system, we used an antibody that recognizes LAMP-1, a protein associated with the lysosome membrane. As shown in Fig. 3, RPE1 cells lacking EHMT1 showed a higher intensity of LAMP-1 labelling, as well as an increase in the size of the lysosomes, which appeared scattered throughout the cell cytoplasm under these conditions. The same phenotype was observed after EHMT1 reduction in HeLa cells (Supplementary Fig. S2). These findings may indicate that EHMT1 has a relevant role in the functioning of the lysosomal system. 3.1.3. EHMT1 role in cytoskeleton organization Cytoskeleton dynamics plays an essential role in synaptic plasticity, allowing the rapid structural changes required in dendritic spines, the main sites of excitatory synaptic input. Some of the mechanisms that determine these synaptic changes are beginning to be uncovered and seem to involve disassembly of adhesion molecules, the breakdown of actin filaments and trafficking of AMPA receptors [29]. Although the role of EHMT1 in dendritic arborization has been described [10], its possible function in the dynamics of the cytoskeleton is unknown. Thus, using specific markers of the different elements of this structure, the role of EHMT1 in the organization of the cytoskeleton was examined. Vimentin labelling, a constituent protein of the intermediate filament network, elements with structural support function, did not reveal significant differences between control cells and cells with decreased levels of EHMT1 (Supplementary Fig. S3A). No apparent differences were also Fig. 3. Decreased EHMT1 leads to lysosomes accumulation. (a) RPE1 cells transfected with the indicated siRNAs were labelled for LAMP-1 (green) and EHMT1 (red). Nuclei were stained with DAPI, and the image overlay is shown in Merge. Bars, 25 μ m, or 7.5 μ m in images of higher magnification. (b) Quantification of immunofluorescence experiments shown in a. LAMP-1 vesicles intensity was analyzed in a total number of cells per condition ≥70. (c) Western blot analysis of EHMT1, LAMP-1, α -tubulin and GAPDH in RPE1 cells after transfection with the indicated siRNAs. (d) Quantification of EHMT1 and LAMP-1 levels, normalized with respect to GAPDH expression. Values are the average and SEM from three independent experiments. Statistical significance was calculated using a t-test (*p <0.05, **p < 0.01, ***p <0.001, ****p <0.0001, ns, not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 6 Fig. 4. Effect of a reduction in EHMT1 levels in the cytoskeleton structure. (a, c) Seventy-two hours after transfection of RPE1 cells with the indicated siRNAs, cells were fixed and processed for immunofluorescence. Antibodies against the indicated proteins were used. Nuclei were stained with DAPI. The image overlay is shown in Merge. Bars, 7.5 μ m. (b, d) Quantification of vinculin (b) and β-catenin (d) intensity levels from immunofluorescence experiments. Total number of scored cells per condition ≥100. (e) RPE1 cells transfected with the indicated siRNAs were analyzed by western blot for the indicated proteins. (f) Quantification of EHMT1, vinculin and β-catenin levels, normalized with respect to GAPDH expression. Values are the average and SEM from three independent experiments. Statistical significance was calculated using a t-test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ns, not significant). L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 7 observed in the organization of the microtubule cytoskeleton, visualized through α -tubulin labelling (Supplementary Fig. S3B). However, when we examined the distribution of vinculin, an actin-binding protein that plays an essential role in the establishment of focal junctions between cells and the extracellular matrix mediated by actin filaments [32], an increase in the number and extension of these junctions under EHMT1 depletion was observed, which could indicate greater adhesion of cells to the matrix (Fig. 4A). Similarly, labelling of β-catenin, a protein involved in the establishment of cell-cell junctions in which the actin cytoskeleton intervenes, showed an increase in the intercellular contact surface (Fig. 4B). Taken together, these results suggest that after EHMT1 reduction, cells bind more strongly to each other and to the extracellular matrix, which could have important implications for processes such as cell migration. These findings are consistent with the observation that after EHMT reduction, the detachment of cells from culture plates requires more time after trypsin treatment. 3.1.4. EHMT1 role in centrosome functioning Defective centrosome function and DNA repair response pathways are the origin of different disorders of intellectual disability in which neurogenesis is affected [33]. Although a role for EHMT1 in DNA repair has been described [34,35], its possible implication in centrosome function has not been explored. The centrosome is the cellular structure that acts as the main center for organizing microtubules been also involved in other essential processes such as cell cycle progression, polarity establishment, migration, mitosis, and ciliogenesis. Thus, in addition to functioning as a microtubule nucleation center, the centrosome acts as a kind of multifunctional platform for numerous signaling processes [36]. Analysis of pericentrin, a protein that acts as a kind of scaffold that recruits different proteins from the pericentriolar material, including γ-tubulin, showed that under EHMT1 reduction, there is an increase in pericentrin and centriolar satellites detection (Fig. 5 and Supplementary Fig. S4), which therefore may indicate a role for EHMT1 in centrosome functioning. Centriolar satellites are microscopically visible structures that appear as dots around the centrosome and contain Fig. 5. Effect of downregulation of EHMT1 in the centrosome. (a) RPE1 cells transfected with the indicated siRNAs were fixed and processed by immunofluorescence after 72 h. Pericentrin labelling is shown in green, and EHMT1 labelling is shown in red. Nuclei were stained with DAPI. The image overlay is shown in Merge. Bars, 7.5 μ m. (b) Quantification of immunofluorescence experiments shown in a. Total number of scored cells per condition ≥60. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 6. EHMT1 is required for migration in RPE1 cells. (a) Cells transfected with the indicated siRNAs were cultured 24 h after transfection on culture inserts (0 h), which were removed 24 h later (24 h). Phase-contrast images of wounds at different locations are displayed. (b) Quantification of migration assays shown in (a). Cells present in the wound area of four different fields were counted for each condition. Values are the average and SEM from three independent experiments. Statistical significance was calculated using a t-test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ns, not significant). L. Iglesias-Ortega et al. Cellular Signalling 108 (2023) 110734 8 numerous proteins involved in centrosome functioning that play an essential role in processes such as ciliogenesis or neurogenesis. For example, the complex formed by the DISC1, BBS4, and PCM1 proteins, present in centriolar satellites, has been described as essential for cell migration during neurogenesis [36], recognized as one of the general cellular mechanisms underlying the pathophysiology of intellectual disability. It is also known that defects in the microtubule nucleation process in the Golgi apparatus, which acts as a secondary microtubule organizing center in cells, lead to a microtubule nucleation process depending on these centriolar structures [37,38]. In this situation, the pericentrin label appears as dotted, similar to the distribution observed after EHMT1 reduction, so the collapsed Golgi observed in these cells could be potentially related to the loss of its microtubule nucleation capacity. Additional experiments will be necessary to corroborate these hypotheses and elucidate the molecular mechanisms responsible for the observed phenotypes. 3.2. EHMT1 is required for RPE1 cells migration Defects in migration are often associated with intellectual disability. To study the effect of EHMT1 downregulation on this key process in neurogenesis, we performed wound migration assays. Interestingly, under EHMT1 depletion, cell migration was reduced by 75% compared to control conditions (Fig. 6). Although we cannot rule out that the migration phenotype observed in EHMT1 depleted cells is partly due to the role of EHMT1 in cell cycle progression, the timing of wound assays is predictably insufficient for a large reduction in cell proliferation. Therefore, the findings may indicate a specific role for EHMT1 in cell migration. We propose that this clear phenotype, which is easy to measure and quantify in classical migration or wound closure assays, could be used as a biomarker for a subsequent study of the effect of candidate molecules for the treatment of Kleefstra patients. 4. Conclusions In this study, we have identified different cellular alterations associated with the reduction of EHMT1 expression. We consider that the described phenotypes could open new possibilities to understand the functional impact of the haploinsufficiency of EHMT1 in Kleefstra syndrome. Additional studies will be necessary to investigate the specific molecular mechanisms by which the decrease in EHMT1 activity leads to them. Transcriptomic analysis in RPE1 cells under EHMT1 depletion could help identify gene disruptions that could be potentially linked to the observed phenotypes in these cells. Additional studies will also be essential to confirm whether the specific alterations observed in the cell lines used in this work are also present in cells of affected people. For that, the use of iPS cells derived from patients could be a useful option. Increased knowledge of the molecular and cellular basis of different neurodevelopmental disorders has enabled, in studies with model organisms, the rescue of at least part of the observed neurological defects [29,39]. These findings offer promising perspectives for possible therapy to ameliorate cognitive deficits that have been considered for a long time irreversible. A rapidly growing number of epigenetic regulators have been associated with cognitive disorders, most of which appear to show regulatory crosstalk. Helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in Kleefstra syndrome could be relevant for understanding and future treatment, not only of this syndrome, but also of other neurodevelopmental conditions that could share common deregulated cellular pathways. Supplementary data to this article can be found online at https://doi. org/10.1016/j.cellsig.2023.110734. Author contributions L.I.-O., C.M.-F., P.D.-G., and S.R. shared the experimental work. S.J.- G. and S.R. supervised the work. S.R. conceived and supervised the project and wrote the manuscript. Funding This study was funded by the University of Seville (Ayudas a Proyectos Precompetitivos del VI Plan Propio de Investigaci´ on granted to S. Rivero). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability No data was used for the research described in the article. References [1] M. Fuentes-Ramos, M. Alaiz-Noya, Scale Bars represent co,“Transcriptome and epigenome analysis of engram cells: Next-generation sequencing technologies in memory research”, Neurosci. Biobehav. Rev. 127 (2021) 865–875, https://doi.org/ 10.1016/J.NEUBIOREV.2021.06.010. [2] M. 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