Biology subjects

Geijsen, N.

Publications and source records attributed to Geijsen, N..

8 recordsLinked to original sources

bioRxiv2026

Sex-specific DNA methylation in adult skeletal muscle

DNA methylation is a key epigenetic mechanism influencing gene regulation and cellular identity. In skeletal muscle, methylation contributes to fiber-type specification, metabolic programming, and satellite cell function, with evidence of sex-specific differences. Here, we investigated whether spatial regionalization of gene expression along the proximal-distal axis of the tibialis anterior (TA) is mirrored by corresponding patterns of DNA methylation. Using MeDseq on TA sections from muscles previously analyzed by spatial transcriptomics, we profiled methylation across transcriptional start sites (TSS), gene bodies, and regulatory elements. Despite robust spatial differences in transcriptomes, methylation patterns were largely uniform along the proximal-distal axis, indicating that DNA methylation does not underlie regional gene expression in adult TA muscle. In contrast, sex emerged as the primary determinant of methylation variation. Male muscles exhibited widespread hypermethylation at TSS, gene-bodies and regulatory regions, corresponding with sex-specific transcriptional programs, including glycolytic fiber enrichment in males and oxidative fiber markers in females. Notably, chromatin- and methylation-associated regulators such as Setd7, Gsk3a, and Bmyc were upregulated in males, suggesting mechanisms linking transcriptional control to epigenetic state. These findings highlight that while spatial gene expression is transcriptionally driven, sex-specific epigenetic programs dominate adult skeletal muscle, underscoring the need to consider sex in multi-omic studies of muscle biology.

molecular biology↗

bioRxiv2023

3D printed magneto-active microfiber scaffolds for remote stimulation of 3D in vitro skeletal muscle models

Tunable culture platforms that guide cellular organization and mechanically stimulate skeletal muscle development are still unavailable due to limitations in biocompatibility and actuation triggered without contact. This study reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a thermoplastic/graphene/iron oxide composite. In situ deposition of iron oxide nanoparticles on oxidized graphene yielded homogeneously dispersed magnetic particles with sizes above 0.5 m and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with very high magnetic filler content (up to 10 wt.%) were obtained and successfully processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material. Importantly, the hexagonal microstructures displayed elastic deformability under tension, mitigating the mechanical limitations due to high filler content. External magnetic fields below 300 mT were sufficient to trigger out-of-plane reversible deformation leading to effective end-to-end length decrease up to 17%. Moreover, C2C12 myoblast culture on 3D Matrigel/collagen/MEW scaffolds showed that the presence of magnetic particles in the scaffolds did not significantly affect viability after 8 days with respect to scaffolds without magnetic filler. Importantly, in vitro culture demonstrated that myoblasts underwent differentiation at similar rates regardless of the presence of magnetic filler. Overall, these innovative microfiber scaffolds were proven as a magnetically deformable platform suitable for dynamic culture of skeletal muscle with potential for in vitro disease modeling.

bioengineering↗

bioRxiv2023

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

CRISPR-Cas13 exclusively targets RNA. In prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. To investigate collateral cleavage by Cas13 in eukaryotic cells, we first compared various Cas13 orthologs and found that specifically LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage started within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulated genes associated with stress and innate immune response, ultimately leading to apoptotic cell death. This enabled us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, we used Nanopore sequencing to explore the identity of collaterally cleaved RNAs, the nucleotide position at which they are cleaved, and the temporal dynamics of collateral RNA cleavage. This revealed that LbuCas13a activation leads to rapid and global cleavage of cytoplasmic RNAs at specific nucleotide positions. In conclusion, we here report that LbuCas13a has high collateral activity in human cells and describe the temporal dynamics of the collateral RNA cleavage, the cellular responses ultimately leading to apoptosis, how this can be exploited as a cell elimination tool, and the collateral cleavage preferences of LbuCas13a.

molecular biology↗

bioRxiv2022

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

molecular biology↗

bioRxiv2021

DUX4 induces a homogeneous sequence of molecular changes, culminating in the activation of a stem-cell-like transcriptional network and induction of apoptosis in somatic cells

Facioscapulohumeral muscular dystrophy (FSHD) is a muscle degenerative disease that disproportionally affects the muscles of the face, shoulder girdle and upper arms. FSHD is caused by the misexpression of Double Homeobox 4 (DUX4), a transcription factor that is normally expressed during early embryonic development. Ectopic expression of DUX4 in somatic cells is cytotoxic and leads to rapid apoptosis. To elucidate the mechanism by which DUX4 induces apoptosis, we determined the temporal transcriptional changes induced by Dux4 at the single-cell level. We observed that induction of DUX4 expression induces a non-random, consecutive sequence of transcriptional changes. DUX4 homogenously induces the activation of a stem cell signature and activates a network of transcription factors that is typically expressed during early embryogenesis and in pluripotent stem cells. Ultimately, these transcriptional changes trigger the induction of apoptosis, suggesting that the induction of this early stemness program is incompatible with a somatic cell program. Our findings shed new light on the timing and dynamic of DUX4-mediated transcriptional reprogramming and may help elucidate why the DUX4 stemness program is required during early embryogenesis, but incompatible with somatic cell viability.

molecular biology↗

bioRxiv2020

A genome-wide CRISPR/Cas phenotypic screen formodulators of DUX4 cytotoxicity reveals screen complications

Facioscapulohumeral muscular dystrophy (FHSD), a fundamentally complex muscle disorder that thus far remains untreatable. As the name implies, FSHD starts in the muscles of the face and shoulder gridle. The main perturbator of the disease is the pioneer transcription factor DUX4, which is misexpressed in affected tissues due to a failure in epigenetic repressive mechanisms. In pursuit of unraveling the underlying mechanism of FSHD and finding potential therapeutic targets or treatment options, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of DUX4 cytotoxicity. We found no key effectors other than DUX4 itself, suggesting treatment efforts in FSHD should be directed towards its direct modulation. The screen did however reveal some rare and unexpected Cas9-induced genomic events, that may provide important considerations for planning future CRISPR/Cas9 knock-out screens.

molecular biology↗

bioRxiv2019

Dynamics of Meiotic Sex Chromosome Inactivation and Pachytene Activation in Mice Spermatogenesis

During germ cell development, cells undergo a drastic switch from mitosis to meiosis to form haploid germ cells. Sequencing and computational technologies now allow studying development at the single-cell level. Here we developed a multiplexed trajectory reconstruction to create a high-resolution developmental map of spermatogonia and prophase-I spermatocytes from testes of a Dazl-GFP reporter mouse. We identified three main transitions in the meiotic prophase-I: meiotic entry, the meiotic sex chromosome inactivation (MSCI), and concomitant pachytene activation. We validated the key features of these transitions in vivo using single molecule FISH. Focusing on MSCI, we found that 34% of sex chromosomal genes are induced shortly before MSCI, that silencing time is diverse and correlates with specific gene functions. These highlight a previously underappreciated level of regulation of MSCI. Finally, we found that spermatozoal genes in pachytene are activated in a temporal pattern reflecting the future anatomic and functional order of the sperm cell. Altogether we highlighted how precise and sequential changes in gene expression regulate cellular states in meiotic prophase-I.

genomics↗

bioRxiv2019

Polar-like trophoblast stem cells form an embryonic-abembryonic axis in blastoids.

The early mammalian conceptus (blastocyst) comprises an outer trophoblast globe that forms an axis originating from the inner embryonic cells. From the mouse conceptus, Trophoblast stem cells (TSCs) are derived, which are in vitro analogues of early trophoblasts. Here, we show that TSCs contain plastic subpopulations reflecting developmental states ranging from pre- to post-implantation trophoblasts. However, upon exposure to a specific combination of embryonic inductive signals, TSCs globally acquire properties of pre-implantation polar trophoblasts (gene expression, self-renewal) juxtaposing the inner embryonic cells, and an enhanced, homogeneous epithelial phenotype. These lines of polar-like TSCs (pTSCs) represent a transcriptionally earlier state that more efficiently forms blastoids, whose inner embryonic cells then induce the patterning of gene expression along the embryonic-abembryonic axis. Altogether, delineating the requirements and properties of polar trophoblasts and blastocyst axis formation in vitro provides a foundation for the precise description and dissection of early development.

developmental biology↗