# Transient pharmacological inhibition of SUMOylation during pregnancy induces craniofacial malformations in offspring mice

Jorge Mata-Garrido, Isabella Zafferri, Alice Nordlinger, Yann Loe-Mie, Anne Dejean, Jack-Christophe Cossec

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## To cite this version:

Jorge Mata-Garrido, Isabella Zafferri, Alice Nordlinger, Yann Loe-Mie, Anne Dejean, et al. Transient pharmacological inhibition of SUMOylation during pregnancy induces craniofacial malformations in offspring mice. European Journal of Cell Biology, 2025, 104 (2), pp.151480. 10.1016/j.ejcb.2025.151480. pasteur-05059934

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# Transient pharmacological inhibition of SUMOylation during pregnancy induces craniofacial malformations in offspring mice

Jorge Mata-Garrido<sup>a,b,*‚1</sup>, Isabella Zafferri<sup>a,b‚2</sup>, Alice Nordlinger<sup>a,b</sup>, Yann Loe-Mie<sup>a,b,c</sup>, Anne Dejean<sup>a,b</sup>, Jack-Christophe Cossec<sup>a,b,*‚1‚3</sup>

## ARTICLE INFO

**Keywords:**

Craniofacial syndromes

SUMOylation

Embryonic development

Epigenetics

Cutis aplasia

UBA2-related syndrome

Neural Crest

**Chemical compounds studied in this article:**
TAK981

## ABSTRACT

Cell identity plays a pivotal role in embryo development, guiding the process of cellular differentiation essential for tissue and organ formation. Post-translational modification by the ubiquitin-related SUMO protein acts as a chromatin barrier to cell fate conversions. While SUMOylation deficiency is incompatible with mammalian embryonic development, haploinsufficiency for the SUMOylation machinery's E1 enzyme, UBA2, leads to various phenotypic traits in humans, including craniofacial malformations and aplasia cutis congenita. To investigate SUMO's role in organogenesis, SUMOylation was transiently suppressed using a specific pharmacological inhibitor, TAK981, administered during the early post-implantation embryo stage. A high-concentration injection led to embryonic lethality associated with epigenetic scars and alterations in nuclear and nucleolar integrity observed in treated embryo-derived fibroblasts. Lower-concentration injections resulted in viable mice with craniofacial deformities often accompanied by hydrocephalus, syndactyly and an aplasia cutis-like phenotype. Transcriptomic analysis revealed the repression of genes involved in neural crest differentiation in the TAK981-treated embryos as well as the overexpression of the *Fgfr* gene family in the adult TAK981 progeny. These genes, expressed in neural crest derivatives, are known for their gain-of-function mutations linked to human craniosynostosis syndromes, suggesting that potential overactivation of the FGF signaling pathway may contribute to the malformations observed in TAK981 progeny. Altogether, disruption of the SUMOylation/deSUMOylation equilibrium during a short embryonic period is sufficient to induce persistent cellular defects and transcriptional alterations, resulting in severe offspring malformations. In conclusion, the SUMO inhibitor TAK981 has teratogenic effects, disrupting normal fetal development and causing congenital disabilities reminiscent of traits observed in UBA2-related syndrome.

## 1. Introduction

The ubiquitin-related SUMO protein, through post-translational modification of chromatin-associated proteins, acts as a key regulator of chromatin function, influencing broad transcriptional programs and playing essential roles in cell homeostasis and stress response (Neyret-Kahn et al., 2013; Cubenas-Potts and Matunis, 2013; Niskanen et al., 2015; Seifert et al., 2015; Decque et al., 2016; Keiten-Schmitz et al., 2021). Specifically, SUMOylation enables the robustness of

protein complexes associated with specific chromatin histone marks, supporting the establishment and maintenance of cell identity (Cossec et al., 2018; Theurillat et al., 2020). Decreasing the level of SUMOylation promotes cell fate changes in models transitioning between pluripotent, totipotent, differentiating, or transdifferentiating states, thereby positioning SUMO as a general guardian of cell identity (Cossec et al., 2018; Borkent et al., 2016; Zhao et al., 2022; Cossec et al., 2023; Baik et al., 2018).

The SUMOylation cascade pathway involves three main steps:

<footnote><sup>a</sup> Institut Pasteur, Université Paris Cité, Nuclear Organization and Oncogenesis Unit, Paris F-75015, France</footnote>

<footnote><sup>b</sup> INSERM, U993, Paris F-75015, France</footnote>

<footnote><sup>c</sup> Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics HUB, Paris F-75015, France</footnote>

<footnote>* Corresponding author at: Institut Pasteur, Université Paris Cité, Nuclear Organization and Oncogenesis Unit, Paris F-75015, France.
E-mail addresses: jorge.mata-garrido@pasteur.fr (J. Mata-Garrido), jack-christophe.cossec@pasteur.fr (J.-C. Cossec).</footnote>

<footnote n="1">¹ Co-senior authors</footnote>

<footnote n="2">² Current address: General pathology laboratory. Department of Clinical Biomedical Sciences. University of Milan. Via G. B. Grassi 74, 20157 Milan</footnote>

<footnote n="3">³ Lead contact.</footnote>
activation, conjugation, and ligation (Vertegaal, 2022). Initially, the main SUMO proteins (SUMO1, SUMO2, SUMO3) are activated by a single E1 enzyme (SAE1/UBA2 heterodimer), then transferred to a single E2 enzyme (UBC9), and finally attached to target proteins by E3 ligases. Additionally, SUMOylation is reversible, with SUMO being removed by SUMO-specific proteases (SENPs), allowing for dynamic control of protein function. During embryonic development, a delicate equilibrium between SUMOylation and deSUMOylation processes is crucial. This balance is underscored by the significant defects observed in patients with partial loss-of-function in the UBA2 gene, including developmental delay, short stature, craniofacial features, ophthalmologic problems, aplasia cutis congenita and ectrodactyly (Marble et al., 2017; Aerden et al., 2020; Wang et al., 2020; Schnur et al., 2021).

Animal models have demonstrated the significant role of SUMOylation during development. Ubc9-deficient mice exhibit abnormalities in nuclear organization and chromosome segregation and show embryonic lethality in the early post-implantation stage (Nacerddine et al., 2005). SUMO orthologues play distinct roles in embryogenesis, SUMO2, in particular, is crucial due to its higher expression levels during early embryonic stages. Null mutant mice lacking SUMO2 exhibit embryonic lethality at stage E10.5, characterized by severe growth retardation, reduced cell proliferation, and increased cell death (Wang et al., 2014). Conversely, SUMO1 or SUMO3 deficiency does not lead to embryonic lethality, suggesting possible functional compensation by other SUMO isoforms. The absence of Pias1, a SUMO E3 ligase, results in impaired embryonic development between stages E10.5 and E12.5 in mice (Constanzo et al., 2016). Additionally, overexpression of the E3 ligase Pias4 in mouse zygotes inhibits zygotic genome activation and compromises early embryo development (Yan et al., 2019). Furthermore, knockout mice lacking the SUMO proteases Senp1 or Senp2 display mid-gestational embryonic lethality (Cheng et al., 2007; Chiu et al., 2008). Whereas loss of Senp1 results in defects in erythropoiesis, lack of Senp2 affects cell cycle progression during mouse trophoblast development, disturbing the p53/Mdm2 pathway and impacting the expansion and maturation of trophoblast progenitors.

Whereas most knockout models for enzymes of the SUMOylation machinery show embryonic lethality, haploinsufficiency models, such as the reduction by half in heterozygous *Ubc9+/-* mice, display very mild phenotypes and thus do not recapitulate the phenotypic traits observed in humans (Demarque et al., 2011; López et al., 2020). To better understand the role of SUMO during embryonic development and explore the safety of SUMOylation inhibitors for potential use in medical settings, we established a novel murine model of hypoSUMOylation by subjecting pregnant females to waves of SUMOylation inhibition. This was achieved through administration of TAK981, a first-in-class SUMOylation inhibitor of the SUMO E1 enzyme that is currently studied in clinical trials in cancer patients (Lightcap et al., 2021), aiming to assess its potential long-term effects in new-born mice. Successive injections of TAK981 in pregnant females led to the emergence of two significant phenotypes in their progeny: 1) high lethality before or shortly after birth, potentially caused by defects in nuclear integrity and alterations in chromatin domain organization; and 2), in viable offspring, a decrease in body weight and size accompanied by mild to severe craniofacial malformations and hydrocephalus, along with hand malformations and blindness. This work highlights the critical role of SUMOylation balance in early development and its enduring effects on the physiology of adult mice, mimicking specific abnormalities observed in patients deficient in the E1 enzyme of the SUMOylation cycle (Marble et al., 2017; Aerden et al., 2020; Wang et al., 2020; Schnur et al., 2021).

# 2. Materials and methods

## 2.1. TAK981 preparation

The compound TAK981 (Subasumstat, MedChemExpress) was prepared in the corresponding vehicle HPBCD [(2-Hydroxypropyl)-

β-cyclodextrin, Sigma-Merck] following manufacturer's instructions. More precisely, the desired amount of TAK981 compound was first dissolved in 40 % HPBCD solution before adjusting pH at 4 and finally completed by dH₂O to reach a final concentration of TAK981 at 5 mg/ml. The vehicle solution used for control condition was prepared from the 40 % HPBCD solution diluted 1:2 in dH₂O. Both TAK981 and vehicle solutions were filtered in 0.2 µm filter before injections in animals.

## 2.2. Animals

All animal experiments were conducted under animal protocols approved by the Ethical Committee of Pasteur Institute and the French Ministry of Research (APAFiS n°44872). C57BL/6 male and female mice were used, and all bred at the Pasteur Institute animal facility under specific pathogen-free conditions. Female mice aged 8-10 weeks were matted and administered intraperitoneally with vehicle (20 % HPBCD diluted in dH₂O) at day E6.5, E7.5 and E8.5 of pregnancy, or 25 mg/kg TAK981 on day E6.5, or 15 mg/kg TAK981 on days E6.5, E7.5 and E8.5. For the survival experiments, mice were sacrificed when reaching the humane endpoints as defined by the Pasteur Ethical Committee. The animals were weighed with a digital rodent balance. For body size and interorbital distance measurements, the animals were immobilized and measured using an electronic caliper. Embryos were extracted at day E12.5 of pregnancy for subsequent isolation and culture of mouse embryonic fibroblasts (MEFs).

## 2.3. Mouse embryonic fibroblast (MEFs) isolation and culture

Pregnant mice injected with HPBCD or 1 dose TAK981 at day E6.5 were sacrificed at day E12.5 of gestation. Embryos were collected in cold PBS, where the placenta and yolk sac were removed. The embryonic cells were then mechanically disaggregated and incubated for 15 min in 0.25 % trypsin-EDTA (Life Technologies), in agitation at room temperature. Digestion was stopped with DMEM supplemented with 10 % FBS and centrifuged for 5 minutes at 1000 rpm at room temperature. The cell pellet was resuspended and cultured in DMEM supplemented with 10 % FBS and penicillin/streptomycin under standard conditions 37°C 5 % CO₂ for 3-5 passages maximum.

## 2.4. Treatment of MEFs with TAK981

To mimic the protocol performed *in vivo*, MEFs were treated *in vitro* with TAK981 at 0.1 µM or 0.25 µM concentration once for 24 hours and analyzed 6 days after drug removal. As a control, an equivalent volume of 20 % HPBCD was diluted in culture medium.

## 2.5. Immunofluorescence and microscopy

MEFs were grown on cover slides to reach 70-80 % confluence. Then, they were fixed with 3.7 % PFA for 10 min at room temperature, permeabilized 15 min with 0.5 % Triton X-100 at room temperature and washed 3 times 5 min with 0.05 % Tween-20. Anti-5-Methylcytosine (BIMECY-0100, Eurogentec), anti-H3K9me3 (ab8898, Abcam), anti-LaminA/C (4777, Cell Signaling Technology), anti-nucleolin (10556-1-AP, Proteintech), anti-SUMO1 (ab32058, Abcam), anti-SUMO2/3 (ab3742, Abcam) were used as primary antibodies at a dilution of 1:200. Nuclei were stained using 4, 6-diamidino-2-phenylindole (DAPI, 62248, Thermo Fisher). Some cells were also counterstained with propidium iodide (P1304MP, Invitrogen) and phalloidin Alexa Fluor 488 (A12379, Invitrogen).

Microscopy images were obtained with a ZEISS Apotome.2 (Zeiss, Germany), structured illumination microscope, using a 63 × oil (1.4 NA) objective. To avoid overlapping signals, images were obtained by sequential excitation at 488 and 594 nm in order to detect A488 and A594 check, respectively. Images were processed using ZEISS ZEN lite software.
## 2.6. RNA Extraction and Real-Time RT-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions, treated with DNase (DNAse I at concentration of 2 u/µg of RNA), was incubated at 37°C for 15 min, following manufacturer's instructions and quantified using a spectrophotometer (Nanodrop Technologies ND-1000). 1 µg of total RNA was used to synthesize cDNA using High Capacity cDNA Reverse Transcription kit (Thermo Fisher). qPCR was performed using the CFX96 real Time System Thermal Cycler (Bio-Rad). The expression levels of the mRNA candidates were determined by qPCR using gene-specific SYBR Green PCR Mastermix (Applied Biosystems)-based primers (0.4 µM). The primers are listed in Supplementary Table 1, with beta-actin used as a housekeeping gene to normalize the expression of the genes of interest. Analyzed gene expression was portrayed as mean ± SD.

## 2.7. RNA Extraction and RNA-seq

Total RNA was extracted from control and Tak-progeny embryos, collected at day E13.5 (n = 6 for control and n = 12 for Tak-progeny) by guanidinium thiocyanate-phenol-chloroform extraction and DNAse treatment. Briefly, RNA was extracted and purified using a TRIzol-based protocol followed by acidic phenol/chloroform purification and DNase treatment to ensure RNA integrity and purity. Samples were homogenized in TRIzol (Invitrogen), followed by chloroform extraction and centrifugation to isolate the aqueous phase. RNA precipitation was achieved with isopropanol, followed by ethanol washes and air drying. Pellets were resuspended in RNase-free water. To enhance RNA quality,

an acidic phenol/chloroform purification was performed twice, including Na-acetate addition and vortexing, with phases separated by centrifugation. Precipitation steps used isopropanol and ethanol, with overnight incubation at -20°C to enhance RNA recovery. DNAse treatment (DNAse I at concentration of 2 u/µg of RNA) was conducted at 37°C for 30 minutes to remove residual DNA, followed by a final acidic phenol/chloroform purification and ethanol precipitation. Final RNA pellets were air-dried and dissolved in RNase-free water, with incubation at 50°C for optimal resuspension, ensuring RNA suitable for downstream applications. The mRNA libraries were performed by BGI Genomics using Hieff NGS Ultima Dual-mode mRNA kit (#12308ES96, Yeasen). The resulting mRNA library was paired-end sequenced (2 × 150 bp) with the sequencer DNBSEQ-G400. The sequencing reads were then aligned to mouse reference genome Mm10 using STAR (2.7.11b) and the annotation from Gencode (vM23). The gene expression matrix was generated using featuresCount from the subread package (v2.0.6) and differentially expressed genes (DEGs) were identified using DESeq2 (v.1.44) (Love et al., 2014). Genes with adjusted P value < 0.05 were considered to be DEGs. All sequencing datasets produced in this study have been deposited at the Gene Expression Omnibus (GEO) and are publicly available (GSE288224).

## 2.8. Protein extraction and analysis by western blot

Animals injected with TAK981 or HPBCD were sacrificed at the indicated times by cervical dislocation. The indicated tissues were collected, in fragments of approximately 5 mm³, transferred to a lysis D matrix tube (MP Biosciences) containing 1 ml of 1X RIPA Lysis Buffer

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Effects of administering the SUMOylation inhibitor TAK981 during gestation on the viability of progeny. (A) Table recapitulating the different protocols used to inject the TAK981 SUMOylation inhibitor into pregnant females at various time points during development and the associated phenotypes observed in the newborns. (B) Schematic representation of the selected protocols for administering the SUMOylation inhibitor TAK981 to pregnant females. (C) Offspring survival with time with 1 maternal dose (25 mg/kg) of TAK981 at day E6.5 of pregnancy (n = 65) or 3 maternal doses (15 mg/kg) TAK981 at days E6.5, E7.5 and E8.5 of pregnancy (n = 90). Control mice were injected with vehicle at days E6.5, E7.5 and E8.5 of pregnancy (control, n = 85). (D) Number of mice born viable per parturient from control- and TAK-progeny females (3 doses of 25 mg/kg). n = 56 control females and n = 28 TAK-treated females, **** adjusted p value < 0.0001, two-tailed t test.</figcaption></figure>
(Millipore) with protease inhibitor cOmplete Mini (Roche) and N-Ethylmaleimide (NEM, Sigma-Merck) and lysed by FastPrep-24 5 G homogenizer (MP Biosciences). Lysates were sonicated for 15 sec and centrifuged at 12,000 rpm at 4°C to collect the supernatant.

MEFs reaching 70-80 % of confluency were washed with PBS and collected in 1 ml of 1X RIPA Lysis Buffer (Millipore) with protease inhibitor cOmplete Mini (Roche) and 20 mM NEM (Sigma-Merck) mechanically lysed by pipetting. Lysates were sonicated for 15 sec and centrifuged at 12,000 rpm at 4°C to collect the supernatant.

Anti-SUMO1 (ab32058, Abcam; 1/500), anti-SUMO2/3 (ab3742, Abcam; 1/500), anti-LaminA/C (4777, Cell Signaling Technology), anti-H3K9me3 (ab8898, Abcam), anti-βactin (20536-1-AP, Proteintech) and anti-GAPDH (ab8245, Abcam; 1/1000) were employed as primary antibodies, followed by the secondary antibody at a 1:10,000 dilution, IRDye 800CW goat anti-rabbit (926-32211, LI-COR) and IRDye 680LT goat-anti-mouse (926-68020, LI-COR). Immunoreactivity was detected with an OdysseyTM Infrared-Imaging System (LI-COR), according to the OdysseyTM Western Blotting Protocol.

## 2.9. Run-On Transcription Assays In Situ

Active transcription sites were labeled by the incorporation of 5'-FU into nascent RNA, as reported (Mata-Garrido et al., 2016). Briefly, MEFs were incubated with 5-FU (Sigma-Merck) at 20 mM from a stock solution at 0.4 M prepared in DMEM. MEFs were fixed after 30 or 60 min of 5'-FU incubation with 3.7 % paraformaldehyde in HPEM buffer (2 × HPEM: Hepes, 60 mM; Pipes, 130 mM; EGTA, 20 mM; and MgCl₂·6 H₂O, 4 mM) containing 0.5 % Triton X-100. The incorporation of 5-FU into nascent RNA was detected with a mouse monoclonal (clone BU-33) anti-BrdU antibody (Sigma-Merck) and then incubated with a secondary antibody coupled with Alexa A488 (Jackson Laboratories).

## 2.10. Histological analysis

Samples were fixed in 10 % neutral buffered formalin for 48 hours followed by EDTA 0.5 M for 10 days and prepared for histopathological evaluations. Samples were embedded in paraffin for 4µm microtome sectioning and stained with hematoxylin and eosin (H&E). Stained slides were evaluated with ZEISS AxioScan.Z1 Digital Slide Scanner and Zen software (Zeiss).

## 2.11. Quantification and statistical analysis

Statistical analyses were performed using GraphPad Prism software. Differences between groups were assayed using a two-sided Student's t test using GraphPad Prism. In all cases, the experimental data were assumed to fulfill t-test requirements (normal distribution and similar variance); in those cases, where the assumption of the t-test was not valid, a nonparametric statistical method was used (Mann-Whitney test). Area and intensity analysis were determined using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij accessed on 1 June 2020).

# 3. Results

## 3.1. SUMOylation inhibition in pregnant mice alters embryo development and offspring survival

Patients with variants in the UBA2 gene exhibit developmental malformations, indicating that SUMOylation is essential during organogenesis following embryo implantation (Marble et al., 2017; Aerden et al., 2020; Wang et al., 2020; Schnur et al., 2021). In addition, complete lack of SUMOylation leads to early embryonic lethality in mice (Nacerddine et al., 2005). To further explore the significance of SUMOylation during development, we opted to hinder SUMO conjugation during the early post-implantation stage in mice, coinciding with

the formation of the three germ layers (ectoderm, mesoderm, endoderm) (Graham and Zernicka-Goetz, 2016).

The drug TAK981, a targeted SUMO E1 inhibitor, has been efficiently used *in vivo* to reduce SUMOylation levels in mice (Lightcap et al., 2021). Our objective was to determine a dosage that inhibits SUMOylation without causing complete lethality in offspring. To this aim, inhibitor concentrations ranging from 7.5 to 25 mg/kg and intraperitoneal injection frequencies of 1-3 doses were tested between E2.5 and E8.5 during early pregnancy development (Fig. 1A). The results showed that initiating SUMOylation inhibition during very early development, at E2.5 or E3.5, resulted in a complete absence of progeny in all five females tested for each condition (Fig. 1A). In contrast, three consecutive doses of 10 mg/kg TAK981 administered at later stages on E6.5, E7.5, and E8.5 of pregnancy or a single 15 mg/kg dose injected on E6.5 resulted in no obvious phenotypic effects in the progeny (Fig. 1A). These results indicate that embryo viability is highly sensitive to variations in SUMOylation during the first days after fertilization, as previously reported (Nacerddine et al., 2005). Increasing the dose to 15 mg/kg TAK981, which corresponds to a commonly used concentration for cancer treatment in mice (Lightcap et al., 2021), with three consecutive doses administered in the same manner (E6.5 to E8.5), led to 41 % offspring survival at the end of the experiment (Fig. 1A-C). This protocol was therefore selected to study the impact of transient SUMOylation suppression during development on the phenotypic traits of the offspring (Fig. 1B). To also investigate a lethal dose for progeny survival, a single high-concentration injection (25 mg/kg) was administered at E6.5 to align with the start of the three-injection protocol (Fig. 1A, B). The single 25 mg/kg dose resulted in 100 % progeny mortality, with no newborn surviving beyond 2 days after delivery (Fig. 1C). Daily monitoring of pregnant females that received three injections of the inhibitor revealed that only 31 % of them delivered pups (adjusted p-value < 2.2e-16, n = 27 control females, n = 41 TAK-treated females), with an average of 4 per litter, whereas all control mice injected with the vehicle gave birth to offspring, with a mean of 9 per litter (Fig. 1D). There was no difference in the sex ratio between the two conditions (Supplementary Fig. 1A). Of note, no overt phenotypes were observed in the injected females post-delivery, including in social behavior, nesting habits, care for newborns, weight, food and water intake, or physical traits. Thus, a temporary decrease in SUMOylation during the post-implantation stage leads to significant embryonic and neonatal mortality shortly after birth.

The kinetics of drug action validated the decrease in modification by SUMO1 and SUMO2/3, mainly through the appearance of free SUMO for the SUMO1 isoform (likely corresponding to an irreversible SUMO-TAK981 adduct (Lightcap et al., 2021), as early as 3 hours post-injection across different organs of the pregnant female, with the inhibition reversing within 24 hours (Supplementary Fig. 1B). Importantly, a significant reduction in SUMOylation, along with the accumulation of free SUMO1, was similarly detected in TAK-progeny embryos compared to control embryos, six hours after the third injection (Supplementary Fig. 1C). These data strongly suggest that the SUMO inhibitor can cross the placental barrier, reaching embryonic tissues, likely explaining the low survival rates observed in the offspring.

## 3.2. Transient SUMOylation inhibition during pregnancy causes persistent chromatin alterations in embryo-derived fibroblasts

A large number of SUMO substrates are closely associated with repressive chromatin complexes as well as with the nuclear envelope, contributing to the formation of extensive protein networks for effective regulation of gene expression (Theurillat et al., 2020; Hendriks and Vertegaal, 2016). To address the high embryonic lethality associated with the transient decrease in SUMOylation induced by injection of TAK981 in pregnant females (Fig. 1C), we hypothesized that genome organization and nuclear integrity may be irreversibly altered in cells of the embryos.
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(caption on next page)
**Fig. 2.** Brief loss of SUMOylation during pregnancy results in lasting chromatin alterations in fibroblasts derived from embryos. (A) Representative immunofluorescence images of DNA methylation patterns in control MEFs and TAK-instructed MEFs (TAK-iMEFs), and quantification of non-canonical DNA methylation patterns. n = 9 clones in total from 3 different mice per condition, with 0/382 nuclei showing non-canonical DNA methylation patterns in control MEFs and 81/404 in TAK-iMEFs, \*\*\* adjusted p-value < 0.0001, binomial test. The dashed line represents the median value. Scale bar: 10 µm. (B) Representative immunofluorescence images of H3K9me3 histone mark in control and TAK-iMEFs. Quantification of the number of H3K9me3 foci in both conditions. n = 158 control MEF nuclei and n = 169 TAK-iMEF nuclei from 9 clones per condition. \*\*\* adjusted p-value < 0.0001, t-test analysis. Scale bar: 25 µm. (C) Representative images of SUMO1 immunostaining in control MEFs and TAK-iMEFs. Arrows indicate heterochromatic foci. Scale bar: 25 µm, scale bar insert: 10 µm. (D) Same as in (C) for SUMO2/3. (E) Percentage of nuclei showing at least one DAPI-positive heterochromatin foci for SUMO1 (left) and SUMO2/3 (right) in control MEFs and TAK-iMEFs. n = 9 clones per condition, with 13/251 showing heterochromatic foci for SUMO1 in control MEF nuclei, 49/280 in TAK-iMEF nuclei, and with 15/243 showing heterochromatic foci for SUMO2/3 in control MEF nuclei and 58/288 in TAK-iMEF nuclei, \*\*\* adjusted p-value < 0.0001, binomial test. The dashed line represents the median value.

We first investigated the repressive epigenetic compartment in mouse embryonic fibroblasts (MEFs) derived from E12.5 embryos. These embryos were derived from pregnant females that had received either a single 25 mg/kg dose of the inhibitor at E6.5 (referred to as TAK-instructed MEFs) or a vehicle (referred to as control MEFs) (Fig. 1A-C). The nuclei of control MEFs exhibited a DNA methylation pattern characterized by small individual foci in 100 % of cells, whereas in TAK-instructed MEFs, approximately 20 % of cells displayed strips of methylated DNA colocalizing with DAPI (Fig. 2A). Moreover, strip-negative TAK-instructed MEFs showed an increase in the number of heterochromatic dots marked by H3K9me3 without global changes in the level of the repressive mark (Fig. 2B, Supplementary Fig. 2A).

Considering that our experimental model is based on the pharmacological induction of transient inhibition of SUMOylation during pregnancy, we wondered whether the observed defects in chromatin reorganization were attributed to the maintenance of low levels of SUMOylation in TAK-instructed MEFs. Immunoblot analysis revealed comparable amount of SUMO conjugates in control and TAK-instructed MEFs (Supplementary Fig. 2B), confirming that hypoSUMOylation is transient and entirely reversible, as observed *in vivo* in pregnant females (Supplementary Fig. 1B). Interestingly, despite complete recovery of normal SUMOylation levels, TAK-instructed MEFs exhibited a distinct sub-cellular localization of SUMO compared to the control counterparts, characterized by an accumulation of both SUMO1 and SUMO2/3 in DAPI-positive heterochromatin foci (Fig. 2C-E).

These findings indicate that MEFs derived from embryos developing in mice subjected to the SUMO inhibitor exhibit aberrant organization of repressive chromatin domains.

### 3.3. Transient SUMOylation inhibition during pregnancy induces long-lasting nuclear alterations in embryo-derived fibroblasts

We then focused on the nuclear envelope, as numerous associated proteins are SUMO substrates, including Lamin A/C whose SUMOylation can cause structural changes resembling laminopathy-type phenotypes (Theurillat et al., 2020; Hendriks and Vertegaal, 2016; Liczmanska et al., 2023). Immunostaining of Lamin A/C revealed nuclear envelope deformations and nuclear blebbing in 19 % and 23 % of TAK-instructed MEFs, compared to 0.2 % and 12 % in control MEFs, respectively (Fig. 3A). These observations are reminiscent of the nuclear dysmorphic features seen in *Ubc9* null mouse embryos (Nacerddine et al., 2005). Furthermore, TAK-instructed MEFs exhibited approximately 4.4 times more micronuclei compared to control MEFs (Fig. 3B). Of note, levels of Lamin A and Lamin C were comparable between both conditions (Supplementary Fig. 2C). In a similar manner, we found a significant increase in the number of multinucleated cells in TAK-instructed MEFs, which displayed larger nuclei and cell sizes (Fig. 3C-F). Although these characteristics are commonly associated with senescent cells (González-Gualda et al., 2021), no difference was observed in the expression of genes specific to this state between the two conditions (Supplementary Fig. 3). Importantly, the defects found in TAK-instructed MEFs were not recapitulated by a transient inhibition of SUMOylation in control MEFs, indicating that the window of hypo-SUMOylation and the embryonic context are critical factors for

triggering alterations of the nuclear structure (Supplementary Fig. 4A, B).

Finally, to probe whether a burst of SUMOylation inhibition in pregnant females may affect global transcription in the developing embryo, we assessed 5 fluorouridine (5-FU) incorporation in MEFs as a marker of newly synthesized RNA. To this end, MEFs were treated with 5-FU for 30 min or 60 min. In control MEFs, both microspeckles and large foci were observed, mainly corresponding to transcription factors for nascent messenger RNA and ribosomal RNA (rRNA), respectively (Fig. 4A, B). Strikingly, TAK-instructed MEFs lacked large foci and instead exhibited a pseudo diffuse staining of the nucleus, (Fig. 4A, B). Furthermore, immunostaining with the nucleolin marker confirmed structural anomalies of the nucleolus, characterized by both increased nucleolar area and number in TAK-instructed MEFs (Fig. 4C). These alterations were associated with a 77 % reduction in the 45S pre-rRNA transcript, indicating a deficiency in rRNA transcription within the nucleolus of TAK-instructed MEFs (Fig. 4D). Such nucleolar restructuring most likely reflects severe nucleolar dysfunction and deficient rRNA transcription.

Altogether, these findings indicate that MEFs derived from embryos developing in mice subjected to the SUMO inhibitor exhibit atypical heterochromatin domains together with drastic defects in nuclear and sub-nuclear architecture, that are likely to contribute to embryonic lethality.

### 3.4. Brief suppression of SUMOylation in pregnant mice leads to craniofacial malformations in offspring

We then analyzed the impact of SUMOylation inhibition during pregnancy in the offspring of mice who had received three doses of 15 mg/kg TAK981 between E6.5 and E8.5. Strikingly, newborns exhibited obvious facial dysmorphisms, including flat nasal structures and a dome-shaped skull, confirmed by tissue staining which revealed abnormal nasal airways (Fig. 5A, B). Since a major phenotype of *UBA2*-related syndrome is cutis aplasia in humans, we closely examined the top of the skull and detected similar phenotypes over the midline cranial sutures (Fig. 5C). These defects may contribute to the 42 % mortality rate among TAK-progeny within the first 2 days post-delivery (Fig. 1C). Furthermore, 17 % of TAK-progeny mice had to be sacrificed within the first month after birth, before weaning and reaching sexual maturity (Fig. 1C). These mice displayed growth retardation, prostration, and hypoactivity. For some, the most pronounced phenotype was congenital hydrocephalus, characterized by a protruding skull containing a significant volume of cerebrospinal fluid (Fig. 5D). This condition likely caused elevated intracranial pressure, as evidenced by structural damage observed upon opening the skull (Fig. 5D). This phenotype was observed only in the TAK-progeny mice (adjusted p-value < 6.4e-15, n = 9/113 in TAK-progeny mice versus n = 0/502 in control mice). Additionally, the hydrocephalic TAK-progeny mice exhibited significant reductions in weight and size, with decreases of nearly 50 % and 24 %, respectively, compared to control mice (Fig. 5E). In conclusion, juvenile morbidity in TAK-progeny mice is, in part, likely caused by cranial malformation leading to hydrocephalus.

A significant proportion (41 %) of TAK-progeny mice showing
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**Fig. 3.** Transient inhibition of SUMOylation in pregnant females causes nuclear abnormalities in fibroblasts derived from embryos. (A) Representative immunofluorescence images of LaminA/C staining in control and TAK-iMEFs with white arrows pointing to nuclear envelop defects and yellow arrows pointing to nuclear blebbing. n = 9 clones from 3 different mothers for control MEFs and n = 10 clones from 3 different mothers for TAK-iMEFs. Quantification with 2/254 nuclei showing envelop abnormalities in control MEFs and 72/390 in TAK-iMEFs, and with 33/254 blebbing nuclei in control MEFs and 91/390 in TAK-iMEFs, \*\*\* adjusted p-value < 0.0001, binomial test. The dashed line represents the median value. Scale bar: 12.5 µm. (B) Representative images of DAPI staining in control MEFs and TAK-iMEFs with arrows pointing to micronuclei. n = 9 clones from 3 different mothers per condition. Quantification with 15/254 cells showing micronuclei in control MEFs and 101/390 in TAK-iMEFs, \*\*\* adjusted p-value < 0.0001, binomial test. The dashed line represents the median value. Scale bar: 10 µm. (C) Representative images of phalloidin and propidium iodide (PI) stainings to detect polynucleated cells (indicated by arrows) in control MEFs and TAK-iMEFs. Localizations of beta-actin (phalloidin) and total RNA (PI) were used to delimit the perimeter of individual cells. Scale bar: 35 µm, scale bar insert: 5 µm. (D-F) Quantification of the number of polynucleated cells (D), nuclear areas (E) and cell areas (F) in control MEFs and TAK-iMEFs. n = 9 clones per condition, with 20/254 polynucleated nuclei in control MEFs and 78/390 in TAK-iMEFs (binomial test) and with analysis of 161 control MEF nuclei and 77 TAK-iMEFs nuclei, 139 control MEFs and 217 TAK-iMEFs (two-tailed t-test). \*\*\* adjusted p-value < 0.0001.

milder phenotypes survived beyond one month (Fig. 1C). In contrast to offspring showing hydrocephalus, these mice displayed normal body size and weight (Supplementary Fig. 5A, B). However, they exhibited distinctive features, notably craniofacial abnormalities, which were less severe than those associated with hydrocephalus. Males had shorter interocular distances compared to their respective controls, a difference not observed in females (Fig. 5F, Supplementary Fig. 5C). Strikingly, 71 % of adult TAK-progeny mice showed opacification in one eye during their growth, in contrast to less than 2 % in control animals (Fig. 5G, H). This phenotypic trait is commonly associated with craniofacial syndromes caused by elevated intracranial pressure (Duan et al., 2021). Additionally, 8 % of the TAK-progeny mice presented syndactyly (Fig. 5I), a characteristic often associated with craniofacial malformations (Al-Qattan, 2019).

To explore the origin of the phenotypic defects associated with transient hypoSUMOylation during embryogenesis, bulk RNA-seq was performed on whole TAK-progeny embryos at day E13.5, five days after the last injection, compared to control embryos. Our focus was to understand the long-lasting effects of the decrease in SUMOylation rather than its acute impact. Comparison of the transcriptomes revealed a significant increase in 350 genes, while 86 genes showed a decrease (Fig. 5J, Supplementary Table 2). Upregulated genes were enriched for ontological terms related to fibrinolysis and blood coagulation, processes that have already been linked to TAK981 injection in adult mice (Lightcap et al., 2021) (Fig. 5K). Interestingly, a significant number of downregulated genes encode transcription factors enriched in the process of neural crest differentiation, including *Lhx1*, *Hes5*, *Ctbp2*, and *Gbx2* (Fig. 5J, L). No other terms related to cell fate change were enriched in the analysis, suggesting that neural crest cells (NCCs) are highly sensitive to variations in SUMOylation, which may impact their fate commitment. Additionally, several components of the Notch signaling pathway were downregulated, including *Dll1*, *Dll3*, and Notch target genes such as *Hes5* (Fig. 5L). These findings indicate a likely repression of the Notch pathway that persists over time, long after the last injection of the SUMOylation inhibitor.

Cranial NCCs are essential for the proper development of the craniofacial skeleton, and defects in their migration, proliferation, or fate, along with their derivatives, are commonly associated with the pathogenesis of developmental craniofacial abnormalities (Siismets and Hatch, 2020). The disorders observed in the TAK-progeny mice evoke human craniofacial syndromes, in which craniofacial anomalies coincide with systemic abnormalities affecting various organ systems. Examples of such syndromes include Apert syndrome, Crouzon syndrome, Pfeiffer syndrome and Muenke syndrome (Yapijakis et al., 2023). These conditions typically result from gain-of-function mutations in genes encoding fibroblast growth factor receptors (FGFRs) such as *FGFR1*, *FGFR2*, and *FGFR3*, which are expressed in neural crest derivatives, leading to distinct facial dysmorphisms, craniosynostosis and related features (Wilkie et al., 1995; Moloney et al., 1997; Vlad Ciurea and Toader, 2009). Interestingly, the transcript levels of *Fgfr* family members were found to be up-regulated in the cerebral cortex and liver of the TAK-progeny (both hydrocephalic mice and adult mice) compared to control mice (Supplementary Fig. 6A-D).

Altogether, these data suggest that brief hypoSUMOylation in pregnant mice induces craniofacial anomalies and related pathologies in offspring, potentially caused by NCC dysfunction during development and the subsequent overexpression of *Fgfr* genes.

## 4. Discussion

In this study, the pharmacological inhibitor TAK981, a highly selective and potent small-molecule inhibitor of SUMOylation, was administered to pregnant mice at the early post-implantation stage, a critical period of embryonic development, to monitor the development of the embryo and newborn mice. This resulted in two major phenotypes depending on the concentration and the number of injections of TAK981: 1/ Injection of a high single dose of inhibitor (25 mg/kg) led to the absence of viable newborns, with no mice surviving at day 2 after delivery. The study of fibroblasts derived from embryos 6 days after maternal injection revealed epigenetic alterations with disorganized DNA methylation and H3K9me3 heterochromatin patterns. Additionally, nuclear integrity was compromised with the occurrence of nuclear blebbing, micro-nuclei and dysfunctional nucleoli. Recent proteomic studies have identified ribosome biogenesis-related proteins as a major group of SUMOylated proteins (Theurillat et al., 2020; Hendriks and Vertegaal, 2016). Maintaining a balanced SUMOylation-deSUMOylation cycle of these factors has been shown to be essential for ribosome functions, which play a key role in regulating protein synthesis, cellular function, cell growth, and proliferation (Finkbeiner et al., 2011; Ryu, 2022). This suggests that the sharp decrease in SUMOylation, albeit transient, caused irreversible nuclear and nucleolar defects that are expected to be detrimental to newborn survival. 2/ Three injections of the inhibitor at a lower concentration (15 mg/kg), whereas being lethal for 42 % of newborn mice by 2 days after delivery, resulted in surviving offspring exhibiting a range of phenotypes. These included reductions in body weight and size, associated with severe hydrocephalus in 29 % of the cases so that mice had to be euthanized. Adult mice that survived beyond 3 weeks of age showed more minor anomalies such as craniofacial malformations, blindness, and syndactyly detected. These phenotypes are commonly associated with syndromic forms of craniosynostosis and related malformations (Katouni et al., 2011), with the most prevalent being Apert, Crouzon, Pfeiffer and Muenke syndromes, which are due to gain of function mutations in *FGFR1*, *FGFR2*, and *FGFR3*. Strikingly, the expression of these genes was found to be significantly increased in both TAK-progeny mice and TAK-instructed MEFs, suggesting a mechanism, whereby a possible overactivation of FGF signaling mediates, at least in part, SUMOylation inhibition-induced craniofacial abnormalities as observed in offspring from TAK981-exposed mothers. In summary, the administration of the pharmacological SUMO inhibitor led to a variety of developmental phenotypic traits, with their severity likely correlating to the dosage received by each embryo. Studying a genetic model that transiently reduces SUMOylation specifically in the embryo would complement our pharmacological approach, allowing us to clearly distinguish the effects of SUMOylation reduction in the embryo from those in the mother.

Multiple lines of evidence suggest that SUMO is involved in
<figure><img src="image_20.png" /><figcaption>**Fig. 4.** Transiently suppressing SUMOylation in pregnant mice alters nucleolar function in embryo-derived fibroblasts. (A, B) Representative images of the incorporation of 5-fluorouridine (5-FU) in nascent transcripts as detected by immunofluorescence in control MEFs and TAK-iMEFs after 30 min of incubation (A) or 60 min of incubation (B), n = 6 clones from 3 different mothers per condition. Scale bars: 25 µm, scale bar inserts: 5 µm. (C) Representative images of nucleolin staining in control MEFs and TAK-iMEFs. n = 9 clones from 3 different mothers per condition. Quantification of the area and number of nucleoli, 645 nucleolar areas in control MEFs, 409 nucleolar areas in TAK-iMEFs, 161 control MEF nuclei, and 130 TAK-iMEF nuclei, \*\* p < 0.01, \*\*\* p < 0.0001, two-tailed t-test. The dashed line represents the mean value. Scale bar: 12.5 µm. (D) Relative expression of the 45S pre-rRNA transcript in control MEFs and TAK-iMEFs. n = 9 clones in total from 3 different mothers per condition, two-tailed t-test, mean ± SD.</figcaption></figure>
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**Fig. 5.** Inhibition of SUMOylation during gestation leads to craniofacial abnormalities in offspring. (A) Representative images of control (left) and TAK-progeny (right) newborn mice. The arrows point to dome-shaped head and facial malformation of treated mice. n = 9 control mice; n = 4 TAK-progeny mice. Scale bar: 1 cm. (B) Hematoxylin & eosin staining of control (left) and TAK-progeny (right) newborn mice. The asterisk points to nasal airways in control mice. Notice the absence of nasal airways in treated mice. Scale bar: 2 mm. (C) Control (left) and TAK-progeny (right) newborn mice. The arrow points to the aplasia cutis-like phenotype observed in treated mice. Scale bar: 1 cm. (D) Views of control (left) and TAK-progeny (right) one-month-old mice showing the full body (top), the meninges (bottom left), and the brain (bottom right). Notice that treated mice exhibit hydrocephalus (arrow), with meninges inflated by cerebrospinal fluid and enlarged brain hemispheres. Scale bar: 1 cm. (E) Body weight (left) and body size (right) on day 22 after birth in control and hydrocephalus mice. Control and hydrocephalus mice were born on the same day from mothers treated or not treated (vehicle) with TAK981, respectively, \*\*\* p < 0.0001, two-tailed t-test. The dashed line represents the mean value. (F) Interorbital distance in control and TAK-progeny males at 5, 6, and 7 weeks after birth. n = 8 control mice and n = 11 TAK-progeny mice, \*\* p < 0.001, \*\*\* p < 0.0001, two-tailed t-test. (G) Side view of TAK-progeny mice showing eye opacification. (H) Graphical representation of the percentage of mice per delivery with eye opacification. 8/502 control mice and 83/113 TAK-progeny mice showed eye opacification from n = 56 control females and n = 28 TAK-treated females. \*\*\* adjusted p-value < 0.0001, binomial test. (I) Representative images of TAK-progeny mice showing syndactyly. 0/502 control mice and 9/113 TAK-progeny mice showed syndactyly \*\*\* adjusted p-value < 0.0001, binomial test. (J) Volcano plot of differentially expressed genes in TAK-progeny embryos compared to control embryos at E13.5 (TAK/Ctrl). The 436 genes that reached statistical significance (P < 0.05) are shown as red dots. Examples of downregulated genes associated with neural crest differentiation are highlighted. (K) Enrichment categories for molecular function, biological process, and pathways of upregulated genes in TAK-progeny embryos at E13.5 compared to control embryos. (L) Same as (K) for downregulated genes.

craniofacial development. Notably, in addition to the UBA2-related syndrome, a translocation breakpoint disrupting SUMO1 was identified in a patient with cleft lip and palate (Alkuraya et al., 2006). Despite the inherent differences between mice and humans, the abnormalities observed in TAK-progeny mice are reminiscent of disorders associated with the human UBA2 genetic syndrome like aplasia-cutis, cranial and hand malformations. These anatomical deformities point to a defect in cranial neural crest commitments (Erickson et al., 2023). Indeed, cranial NCCs, which emerge shortly after implantation around E7.5, give rise to the majority of the head's connective and skeletal structures. Transcriptomic analysis revealed that decreased SUMOylation, triggered by prenatal exposure to TAK981 between E6.5 and E8.5, specifically reduced gene expression related to neural crest differentiation in the embryo at E13.5, potentially altering cranial NCC functions during development. Among the nearly thousand SUMO protein substrates identified so far, transcription factors and chromatin regulators represent the largest class of targets (Hendriks and Vertegaal, 2016). Notably, SUMOylation has been shown to be essential for the activity of key transcription factors involved in NCC development such as Sox9, Sox10, Pax7 and Zic5 (Taylor and LaBonne, 2005; Liu et al., 2013; Luan et al., 2013; Ali et al., 2021). However, the role of the SUMO pathway in NCC homeostasis remains largely unexplored. Specifically, it will be important to clarify the role of SUMOylation of other bona fide SUMO substrates similarly shown to play an important role in this process. These include *Kctd1* and *Kctd15*, whose loss in NCCs result in skull anomalies and cutis aplasia in mice (Raymundo et al., 2023), and mutations in humans are implicated in craniofacial conditions such as scalp-ear-nipple syndrome (Marneros et al., 2013; Miller et al., 2024). More generally, establishing the repertoire of SUMOylated proteins at large in NCCs could be particularly insightful to better understand the role of SUMO in NCC biology, and to assess whether possible neural crest developmental defects may contribute to UBA2-related human syndrome.

Transcriptome analysis also revealed another important point: the decreased expression of genes involved in the Notch signaling pathway in TAK-progeny embryos. Since the analysis was performed on the whole embryo, we cannot confirm that this alteration occurs in NCCs. However, it is important to note that the Notch pathway plays a critical role in the emergence, migration, and differentiation of NCCs in both in vivo and in vitro models (Mead and Yutzey, 2012; Noisa et al., 2014). Interestingly, Adams-Oliver syndrome, a rare human disorder primarily resulting from mutations in genes associated with the NOTCH signaling pathway, including *NOTCH1* and *DLL4*, is characterized by aplasia cutis of the scalp (Stittrich et al., 2014; Meester et al., 2015), a phenotype similar to UBA2-related syndrome. Additional research will be required to determine whether the transient decrease in SUMOylation impacts the NOTCH pathway in NCCs during development.

It has been shown that genetic inhibition of SUMOylation in myeloid cells triggers a potent type I interferon (IFN1) response (Decque et al.,

2016). In line with these findings, TAK981 exhibits a potent anti-tumoral activity, which is dependent on IFN1 signaling (Lightcap et al., 2021; Nakamura et al., 2022; Kumar et al., 2022). A clinical trial combining TAK981 with anti-PD1 is ongoing for the treatment of patients with advanced or metastatic solid tumors (clinicaltrials.gov. Identifier: NCT04381650). Although promising in the context of cancer therapy, the drug's impact on embryonic development has never been documented to the best of our knowledge. Our study describes the teratogenic effects of the drug on embryo physical development when administered to pregnant mice.

## 5. Conclusion

Transient hypoSUMOylation in pregnant females after embryo implantation revealed developmental malformations, including craniofacial defects in both newborns and adults, reminiscent of the human syndrome associated with loss-of-function mutations in the gene encoding the E1 enzyme of the SUMOylation cycle. This pharmacological approach provides a proof of concept for precisely dissecting the role of SUMO at different developmental stages and for monitoring the long-term effects of SUMOylation inhibitors at the molecular, cellular, and whole-organism levels, if these inhibitors were to be used in the clinic for the treatment of cancer and other SUMO-related pathologies.

## Author contributions

J.M.-G and J.-C.C. planned the studies. J.M.-G and I.Z. performed most of the experiments. J.M.-G, I.Z., A.D., and J.-C.C analyzed the data. Y.L.-M. analyzed the RNA-seq data, A.N. contributed to the mice studies. A.D. provided intellectual support and contributed to the writing of the manuscript. J.M.-G and J.-C.C. wrote the paper.

## CRediT authorship contribution statement

**Nordlinger Alice:** Writing – review & editing, Investigation. **Zaf ferri Isabella:** Investigation. **Dejean Anne:** Writing – review & editing, Writing – original draft, Funding acquisition, Formal analysis. **Loe-Mie Yann:** Investigation, Formal analysis. **Mata-Garrido Jorge:** Writing – original draft, Supervision, Project administration, Methodology, Investigation, Conceptualization. **Cossec Jack-Christophe:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Formal analysis, Conceptualization.

## 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
## Acknowledgments

The authors thank Magali Tichit (Histology facility, Institut Pasteur) for processing histological samples and Alexandra Andrieux for writing the bioethics document. This work was supported by grants from European Research Council (AdG SUMIDENTITY), Agence Nationale de la Recherche (ANR-19-CE12-0011-01) and Sjöberg Foundation to A.D.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejcb.2025.151480.

## Data availability

Data will be made available on request.

## References

Aerden, M., Bauters, M., Van Den Bogaert, K., Vermeesch, J.R., Holvoet, M., Plasschaert, F., Devriendt, K., 2020. Genotype-phenotype correlations of UBA2 mutations in patients with ectrodactyly. Eur. J. Med. Genet. 63, 104009. https://doi.org/10.1016/j.ejmg.2020.104009.

Ali, R.G., Bellchambers, H.M., Warr, N., Ahmed, J.N., Barratt, K.S., Neill, K., Diamand, K. E.M., Arkell, R.M., 2021. WNT-responsive SUMOylation of ZIC5 promotes murine neural crest cell development, having multiple effects on transcription. J. Cell Sci. 134, jcs256792. https://doi.org/10.1242/jcs.256792.

Alkuraya, F.S., Saadi, I., Lund, J.J., Turbe-Doan, A., Morton, C.C., Maas, R.L., 2006. SUMO1 Haploinsufficiency Leads to Cleft Lip and Palate. Science 313, 1751. https://doi.org/10.1126/science.1128406.

Al-Qattan, M.M., 2019. A Review of the Genetics and Pathogenesis of Syndactyly in Humans and Experimental Animals: A 3-Step Pathway of Pathogenesis. BioMed. Res. Int. 2019, e9652649. https://doi.org/10.1155/2019/9652649.

Baik, H., Boulanger, M., Hoseini, M., Kowalczyk, J., Zaghdoudi, S., Salem, T., Sarry, J.-E., Hicheri, Y., Cartron, G., Piechaczky, M., Bossis, G., 2018. Targeting the SUMO Pathway Primes All-trans Retinoic Acid-Induced Differentiation of Nonpromyelocytic Acute Myeloid Leukemias. Cancer Res 78, 2601-2613. https://doi.org/10.1158/0008-5472.CAN-17-3361.

Borkent, M., Bennett, B.D., Lackford, B., Bar-Nur, O., Brumbaugh, J., Wang, L., Du, Y., Fargo, D.C., Apostolou, E., Cheloufi, S., Maherali, N., Elledge, S.J., Hu, G., Hochedlinger, K., 2016. A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2. Stem Cell Rep. 6, 704-716. https://doi.org/10.1016/j.stemcr.2016.02.004.

Cheng, J., Kang, X., Zhang, S., Yeh, E.T.H., 2007. SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia. Cell 131, 584-595. https://doi.org/10.1016/j.cell.2007.08.045.

Chiu, S.-Y., Asai, N., Costantini, F., Hsu, W., 2008. SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages. PLoS Biol. 6, e310. https://doi.org/10.1371/journal.pbio.0060310.

Constanzo, J.D., Deng, M., Rindhe, S., Tang, K.-J., Zhang, C.-C., Scaglioni, P.P., 2016. Pias1 is essential for erythroid and vascular development in the mouse embryo. Dev. Biol. 415, 98-110. https://doi.org/10.1016/j.ydbio.2016.04.013.

Cossec, J.-C., Theurillat, I., Chica, C., Búa Aguín, S., Gaume, X., Andrieux, A., Iturbide, A., Jouvion, G., Li, H., Bossis, G., Seeler, J.-S., Torres-Padilla, M.-E., Dejean, A., 2018. SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States. Cell Stem Cell 23, 742-757.e8. https://doi.org/10.1016/j stems.2018.10.001.

Cossec, J.-C., Traboulsi, T., Sart, S., Loe-Mie, Y., Guthmann, M., Hendriks, I.A., Theurillat, I., Nielsen, M.L., Torres-Padilla, M.-E., Baroud, C.N., Dejean, A., 2023. Transient suppression of SUMOylation in embryonic stem cells generates embryonic-like structures. Cell Rep. 42, 112380. https://doi.org/10.1016/j. celrep.2023.112380.

Cubenas-Potts, C., Matunis, M.J., 2013. SUMO: a multifaceted modifier of chromatin structure and function. Dev. Cell 24, 1-12. https://doi.org/10.1016/j. devcel.2012.11.020.

Decque, A., Joffre, O., Magalhaes, J.G., Cossec, J.C., Blecher-Gonen, R., Lapaquette, P., Silvin, A., Manel, N., Joubert, P.E., Seeler, J.S., Albert, M.L., Amit, I., Amigorena, S., Dejean, A., 2016. Sumoylation coordinates the repression of inflammatory and anti-viral gene-expression programs during innate sensing. Nat. Immunol. 17, 140-149. https://doi.org/10.1038/ni.3342.

Demarque, M.D., Nacerddine, K., Neyret-Kahn, H., Andrieux, A., Danenberg, E., Jouvion, G., Bomme, P., Hamard, G., Romagnolo, B., Terris, B., Cumano, A., Barker, N., Clevers, P., Dejean, A., 2011. Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice. Gastroenterology 140, 286-296. https://doi.org/10.1053/j.gastro.2010.1002.

Duan, M., Skoch, J., Pan, B.S., Shah, V., 2021. Neuro-Ophthalmological Manifestations of Craniosynostosis: Current Perspectives. Eye Brain 13, 29-40. https://doi.org/10.2147/EB.S234075.

Erickson, A.G., Kameneva, P., Adameyko, I., 2023. The transcriptional portraits of the neural crest at the individual cell level. Semin. Cell Dev. Biol. 138, 68-80. https://doi.org/10.1016/j semcdb.2022.02.017.

Finkbeiner, E., Haindl, M., Raman, N., Muller, S., 2011. SUMO routes ribosome maturation. Nucleus 2, 527-532. https://doi.org/10.4161/nucl.2.6.17604.

González-Gualda, E., Baker, A.G., Fruk, L., Munoz-Espín, D., 2021. A guide to assessing cellular senescence *in vitro* and *in vivo*. FEBS J. 288, 56-80. https://doi.org/10.1111/ febs.15570.

Graham, S.J.L., Zernicka-Goetz, M., 2016. The Acquisition of Cell Fate in Mouse Development. Current Topics in Developmental Biology. Elsevier, pp. 671-695. https://doi.org/10.1016/bs.ctdb.2015.11.021.

Hendriks, I.A., Vertegaal, A.C., 2016. A comprehensive compilation of SUMO proteomics. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/nrm.2016.81.

K. Katouni, A. Nikolaou, T. Mariolis, V. Protogerou, D. Chrysikos, S. Theofilopoulou, D. Filippou, Syndromic Craniosynostosis: A Comprehensive Review, Cureus 15 (n.d.) e50448. https://doi.org/10.7759/cureus.50448).

Keiten-Schmitz, J., Röder, L., Hornstein, E., Müller-McNicoll, M., Müller, S., 2021. SUMO: Glue or Solvent for Phase-Separated Ribonucleoprotein Complexes and Molecular Condensates? Front. Mol. Biosci. 8, 673038. https://doi.org/10.3389/fmolb.2021.673038.

Kumar, S., Schoonderwoerd, M.J.A., Kroonen, J.S., de Graaf, I.J., Sluijter, M., Ruano, D., González-Prieto, R., Verlaan-de Vries, M., Rip, J., Arens, R., de Miranda, N.F.C.C., Hawinkels, L.J.A.C., van Hall, T., Vertegaal, A.C.O., 2022. Targeting pancreatic cancer by TAK-981: a SUMOylation inhibitor that activates the immune system and blocks cancer cell cycle progression in a preclinical model. Gut 71, 2266-2283. https://doi.org/10.1136/gutjnl-2021-324834.

Liczman ska, M., Tatham, M.H., Mojsa, B., Eguiu-Anta, A., Rojas-Fernandez, A., Ibrahim, A.F.M., Hay, R.T., 2023. SUMO protease SENP6 protects the nucleus from hyperSUMOylation-induced laminopathy-like alterations. Cell Rep. 42, 112960. https://doi.org/10.1016/j.celrep.2023.112960.

Lightcap, E.S., Yu, P., Grossman, S., Song, K., Khattar, M., Xega, K., He, X., Gavin, J.M., Imaichi, H., Garnsey, J.J., Koenig, E., Zhang, H., Lu, Z., Shah, P., Fu, Y., Milhollen, M.A., Hatton, B.A., Riceberg, J., Shinde, V., Li, C., Minissale, J., Yang, X., England, D., Klinghoff, R.A., Langston, S., Galvin, K., Shapiro, G., Pulukuri, S.M., Fuchs, S.Y., Huszar, D., 2021. A small-molecule SUMOylation inhibitor activates antitumor immune responses and potentiates immune therapies in preclinical models. Sci. Transl. Med 13, eaba7791. https://doi.org/10.1126/scitranslmed.aba7791.

Liu, J.A.J., Wu, M.-H., Yan, C.H., Chau, B.K.H., So, H., Ng, A., Chan, A., Cheah, K.S.E., Briscoe, J., Cheung, M., 2013. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling. Proc. Natl. Acad. Sci. U. S. A. 110, 2882-2887. https://doi.org/10.1073/pnas.1211747110.

López, I., Chalatsi, E., Ellenbroek, S.I.J., Andrieux, A., Roux, P.-F., Capbio, J.P., Jouvion, G., van Rheenen, J., Seeler, J.-S., Dejean, A., 2020. An unanticipated tumor-suppressive role of the SUMO pathway in the intestine unveiled by Ubc9 haploinsufficiency. Oncogene 39, 6692-6703. https://doi.org/10.1038/s41388-020-01457-y.

Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. https://doi.org/10.1186/s13059-014-0550-8.

Luan, Z., Liu, Y., Stuhlmiller, T.J., Marquez, J., García-Castro, M.I., 2013. SUMOylation of Pax7 is essential for neural crest and muscle development. Cell. Mol. Life Sci. 70, 1793-1806. https://doi.org/10.1007/s00108-012-1220-1.

Marble, M., Guillen Sacoto, M.J., Chikarmane, R., Gargiulo, D., Juusola, J., 2017. Missense variant in UBA2 associated with aplasia cutis congenita, duane anomaly, hip dysplasia and other anomalies: A possible new disorder involving the SUMOylation pathway. Am. J. Med. Genet. Part A 173, 758-761. https://doi.org/10.1002/ajmgA.38078.

Marneros, A.G., Beck, A.E., Turner, E.H., McMillin, M.J., Edwards, M.J., Field, M., de Macena Sobreira, N.L., Perez, A.B.A., Fortes, J.A.R., Lampe, A.K., Giovannucci Uzielli, M.L., Gordon, C.T., Plessis, G., Le Merrer, M., Amiel, J., Reichenberger, E., Shively, K.M., Cerrato, F., Labow, B.I., Tabor, H.K., Smith, J.D., Shendure, J., Nickerson, D.A., Bamshad, M.J., 2013. Mutations in KCTD1 Cause Scalp-Ear-Nipple Syndrome. Am. J. Hum. Genet 92, 621-626. https://doi.org/10.1016/j. ajhg.2013.03.002.

Mata-Garrido, J., Casafont, I., Tapia, O., Berciano, M.T., Lafarga, M., 2016. Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization. Acta Neuropathol. Commun. 4, 41. https://doi.org/10.1186/s40478-016-0312-9.

Mead, T.J., Yutzey, K.E., 2012. Notch pathway regulation of neural crest cell development in vivo. Dev. Dyn. 241, 376-389. https://doi.org/10.1002/dvdv.23717.

Meester, J.A.N., Southgate, L., Stittrich, A.-B., Venselaar, H., Beekmans, S.J.A., den Hollander, N., Bijlsma, E.K., Helderman-van den Enden, A., Verheij, J.B.G.M., Glusman, G., Roach, J.C., Lehman, A., Patel, M.S., de Vries, B.B.A., Ruivenkamp, C., Itin, P., Prescott, K., Clarke, S., Trembath, R., Zenker, M., Sukalo, M., Van Laer, L., Loeys, B., Wuyts, W., 2015. Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome. Am. J. Hum. Genet. 97, 475-482. https://doi.org/10.1016/j.ajhg.2015.07.015.

Miller, K.A., Walma, D.A.C., Pinkas, D.M., Tooze, R.S., Butfon, J.C., Richardson, W., Manning, C.E., Hunt, A.E., Cros, J., Hartill, V., Parker, M.J., McGowan, S.J., Twigg, S.R.F., Chalk, R., Staunton, D., Johnson, D., Wilkie, A.O.M., Bullock, A.N., 2024. BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome. J. Med. Genet. 61, 490-501. https://doi.org/10.1136/jmgb-2023-109531.
Moloney, D.M., Wall, S.A., Ashworth, G.J., Oldridge, M., Glass, I.A., Francomano, C.A., Muenke, M., Wilkie, A.O., 1997. Prevalence of Pro250Arg mutation of fibroblast growth factor receptor 3 in coronal craniosynostosis. Lancet 349, 1059–1062.
https://doi.org/10.1016/s0140-6736(96)09082-4.

Nacerddine, K., Lehembre, F., Bhaumik, M., Artus, J., Cohen-Tannoudji, M., Babinet, C., Pandolfi, P.P., Dejean, A., 2005. The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice. Dev. Cell 9, 769–779. https://doi.org/10.1016/j.devcel.2005.10.007.

Nakamura, A., Grossman, S., Song, K., Xega, K., Zhang, Y., Cvet, D., Berger, A., Shapiro, G., Huszar, D., 2022. The SUMOylation inhibitor subasumstat potentiates rituximab activity by IFN1-dependent macrophage and NK cell stimulation. Blood 139, 2770–2781. https://doi.org/10.1182/blood.2021014267.

Neyret-Kahn, H., Benhamed, M., Ye, T., Le Gras, S., Cossec, J.C., Lapaquette, P., Bischof, O., Ouspenskaia, M., Dasso, M., Seeler, J., Davidson, I., Dejean, A., 2013. Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation. Genome Res 23, 1563–1579.
https://doi.org/10.1101/gr.154872.113.

Niskanen, E.A., Malinen, M., Sutinen, P., Toropainen, S., Paakinkaho, V., Vihervaara, A., Joutsen, J., Kaikkonen, M.U., Sistonen, L., Palvimo, J.J., 2015. Global SUMOylation on active chromatin is an acute heat stress response restricting transcription. Genome Biol. 16, 153. https://doi.org/10.1186/s13059-015-0717-y.

Noisa, P., Lund, C., Kanduri, K., Lund, R., Lähdesmäki, H., Lahesmaa, R., Lundin, K., Chokechuwattananalert, H., Otonkoski, T., Tuuri, T., Raivio, T., 2014. Notch signaling regulates neural crest differentiation from human pluripotent stem cells. J. Cell Sci., jcs.145755 https://doi.org/10.1242/jcs.145755.

Raymundo, J.R., Zhang, H., Smaldone, G., Zhu, W., Daly, K.E., Glennon, B.J., Pecoraro, G., Salvatore, M., Devine, W.A., Lo, C.W., Vitagliano, L., Marneros, A.G., 2023. KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions, and their impairment causes aplasia cutis. J. Clin. Invest 134, e174138. https://doi.org/10.1172/JCI174138.

Ryu, H.-Y., 2022. SUMO pathway is required for ribosome biogenesis. BMB Rep. 55, 535–540. https://doi.org/10.5483/BMBRep.2022.55.11.130.

Schnur, R.E., Yousaf, S., Liu, J., Chung, W.K., Rhodes, L., Marble, M., Zambrano, R.M., Sobreira, N., Jayakar, P., Pierpont, M.E., Schultz, M.J., Pichurin, P.N., Olson, R.J., Graham, G.E., Osmond, M., Contreras-García, G.A., Campo-Neira, K.A., Peñaloza-Mantilla, C.A., Flage, M., Kuppa, S., Navarro, K., Sacoto, M.J.G., Wentzensen, I.M., Scarano, M.I., Juusola, J., Prada, C.E., Hufnagel, R.B., 2021. UBA2 variants underlie a recognizable syndrome with variable aplasia cutis congenita and ectrodactyly. Genet. Med. 23, 1624–1635. https://doi.org/10.1038/s41436-021-01182-1.

Seifert, A., Schofield, P., Barton, G.J., Hay, R.T., 2015. Proteotoxic stress reprograms the chromatin landscape of SUMO modification. Sci. Signal 8, rs7. https://doi.org/10.1126/scisignal.aaa2213.

Siismets, E.M., Hatch, N.E., 2020. Cranial Neural Crest Cells and Their Role in the Pathogenesis of Craniofacial Anomalies and Coronal Craniosynostosis. JDB 8, 18.
https://doi.org/10.3390/jdb8030018.

Stittrich, A.-B., Lehman, A., Bodian, D.L., Ashworth, J., Zong, Z., Li, H., Lam, P., Khromykh, A., Iyer, R.K., Vockley, J.G., Baveja, R., Silva, E.S., Dixon, J., Leon, E.L., Solomon, B.D., Glusman, G., Niederhuber, J.E., Roach, J.C., Patel, M.S., 2014. Mutations in NOTCH1 Cause Adams-Oliver Syndrome. Am. J. Hum. Genet. 95, 275–284. https://doi.org/10.1016/j.ajhg.2014.07.011.

Taylor, K.M., LaBonne, C., 2005. SoxE Factors Function Equivalently during Neural Crest and Inner Ear Development and Their Activity Is Regulated by SUMOylation. Dev. Cell 9, 593–603. https://doi.org/10.1016/j.devcel.2005.09.016.

Theurillat, I., Hendriks, I.A., Cossec, J.-C., Andrieux, A., Nielsen, M.L., Dejean, A., 2020. Extensive SUMO Modification of Repressive Chromatin Factors Distinguishes Pluripotent from Somatic Cells. Cell Rep. 32, 108146. https://doi.org/10.1016/j.celrep.2020.108146.

Vertegaal, A.C.O., 2022. Signalling mechanisms and cellular functions of SUMO. Nat. Rev. Mol. Cell Biol. 23, 715–731. https://doi.org/10.1038/s41580-022-00500-y.

Vlad Ciurea, A., Toader, C., 2009. Genetics of craniosynostosis: review of the literature.
J. Med Life 2, 5-17.

Wang, L., Wansleeben, C., Zhao, S., Miao, P., Paschen, W., Yang, W., 2014. SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep. 15, 878–885. https://doi.org/10.15252/embr.201438534.

Wang, Y., Dupuis, L., Jobling, R., Kannu, P., 2020. Aplasia cutis congenita associated with a heterozygous loss-of-function UBA2 variant. Br. J. Dermatol. 182, 792–794.
https://doi.org/10.1111/bjd.18594.

Wilkie, A.O., Slaney, S.F., Oldridge, M., Poole, M.D., Ashworth, G.J., Hockley, A.D., Hayward, R.D., David, D.J., Pulleyn, L.J., Rutland, P., 1995. Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat. Genet 9, 165–172. https://doi.org/10.1038/ng0295-165.

Yan, Y.-L., Zhang, C., Hao, J., Wang, X.-L., Ming, J., Mi, L., Na, J., Hu, X., Wang, Y., 2019. DPPA2/4 and SUMO E3 ligase PIAS4 opposingly regulate zygotic transcriptional program. PLoS Biol. 17, e3000324. https://doi.org/10.1371/journal.pbio.3000324.

Yapijakis, C., Pachis, N., Sotiriadou, T., Vaila, C., Michopoulou, V., Vassiliou, S., 2023. Molecular Mechanisms Involved in Craniosynostosis. Vivo 37, 36–46. https://doi.org/10.21873/invivo.13052.

Zhao, X., Hendriks, I.A., Le Gras, S., Ye, T., Ramos-Alonso, L., Nguéa P, A., Lien, G.F., Ghasemi, F., Klungland, A., Jost, B., Enserink, J.M., Nielsen, M.L., Chymkowitch, P., 2022. Waves of sumoylation support transcription dynamics during adipocyte differentiation. Nucleic Acids Res. 50, 1351–1369. https://doi.org/10.1093/nar/gkac027.