Biology subjects

van IJcken, W. F. J.

Publications and source records attributed to van IJcken, W. F. J..

12 recordsLinked to original sources

bioRxiv2026

A role for tubulin in cellular quality control and proteostasis

Microtubules, stiff rods built up from tubulin dimers, form a cytoskeletal network whose structure, behaviour, and function have been extensively investigated, mainly from a mechanical perspective. Here, we describe a role for tubulin in the cellular stress response. We overexpressed tubulin dimers in a controlled fashion in 293F cells. Despite the engagement of autoregulation, a mechanism that degrades tubulin-encoding mRNAs when tubulin levels are high, a surplus of tubulin and microtubules is detected in overexpressing cells. This leads to altered microtubule behaviour, mitotic problems, deregulation of the cell cycle, and replication stress. Surprisingly, we also observe proteostasis defects in tubulin overexpressing cells, which we attribute to mitochondrial stress-related translation attenuation. Conversely, tubulin and microtubules are downregulated as part of the response to oxygen or glutamine deprivation. Together, our data link tubulin levels, and hence autoregulation, to cellular quality control and proteostasis. We propose that competitive interactions with key partners, including the mitochondrial protein import and general translation machinery, underlie the tubulin-mediated control of cellular homeostasis.

cell biology↗

bioRxiv2025

A Novel XACT lncRNA Transcript with Functions Transcending X-Chromosome Inactivation.

X-chromosome inactivation (XCI) is initiated during early mammalian embryogenesis by a long non-coding RNA (lncRNA) XIST, which coats one of the two X-chromosomes and facilitates epigenetic transcriptional silencing. A second, evolutionarily recent primate-specific lncRNA XACT was proposed to antagonize XISTs ability to induce XCI. XACT expression is restricted to pluripotent states and early embryonic stages and coats the active X-chromosome in both females and males. Here, we report a novel XACT transcript expressed in normal and cancerous somatic cells from both the inactive (Xi) and active (Xa) X chromosomes. It coexists with XIST on the Xi without affecting XIST expression or (re)activating X-linked genes inactivated by XCI. During hematopoietic stem cell (HSC) differentiation, XACT is primarily expressed in myeloid progenitors in both sexes. XACT expression is activated in HSCs and peaks in megakaryocyte-erythrocyte progenitor cells (MEPs) before rapidly declining as the MEPs differentiate into megakaryocytes or erythrocytes. By combining CRISPR-based XACT perturbation with epigenomic and transcriptional studies, we revealed the key role of XACT in the self-renewal and differentiation of erythroid progenitors into erythrocytes, by recruiting cis-regulatory proteins and regulating transcription through ETS and AP-1 transcription factors. Furthermore, XACT is expressed in a subset of acute myeloid leukemia (AML) patients, with high levels found in erythroid-megakaryocytic blast cells; suggesting XACTs potential as a marker for AML subtypes. Thus, we identified a novel XACT transcript with a unique expression profile and important roles in normal and cancer cells, revealing XACT lncRNA functions beyond its previously proposed role in XCI during embryogenesis.

genetics↗

bioRxiv2024

The Integrator complex prevents premature neuronal differentiation through global control of transcription elongation.

Transcription pause-release constitutes a key step in the regulation of gene expression. Pathogenic variants in various pausing regulators have been identified in neurodevelopmental disorders, but how transcription pause-release regulates neural development remains largely unknown. Here we show that a patient-specific in-frame deletion in Integrator subunit 8 (INTS8) that prevents association of this subunit with the Integrator complex results in a global increase in nascent transcription and precocious expression of neuronal genes in progenitor cells. Consequently, differentiating neural progenitors fail to sustain a proliferating progenitor cell population during cortical neuronal network formation, resulting in network collapse. We show that attenuation of RNApol2 pause-release by targeted degradation of BRD4 can revert neuronal gene activation and prevent premature progenitor cell loss. Our data demonstrate that a fine-tuned balance between RNApol2 pausing and elongation is crucial for normal neuronal development and suggest transcription pause-release as a druggable target for neurological defects in developmental disorders.

developmental biology↗

bioRxiv2022

Chromatin architecture and cis-regulatory landscape of the DACT2-SMOC2 locus in the developing synovial joint.

BackgroundSynovial joints form in several steps, starting with the formation of an interzone, a condensation of mesenchymal cells at the sites of prospective joints. Despite the identification of multiple factors essential for formation of interzone, little is known about the regulation of their spatio-temporal gene expression during that process in limb development. Here, we investigated the cis- regulatory landscape of the Wnt-modulator encoding genes DACT2 and SMOC2, both expressed in the forming joint interzone. ResultsMechanically collected interzone and phalange samples, respectively, from chick embryos were found to express acknowledged marker genes (GDF5 and MATN1), as well as DACT2 and SMOC2. Using Targeted Chromatin Capture (T2C) we characterized the 3D chromatin structure of a ~3.45 Mb-long region encompassing DACT2 and SMOC2, which revealed differences at sub-TAD level between interzones and phalange. We identified candidate enhancers (CEs) based on H3-histone marks (H3K427ac and H3K4me1) located in close proximity to the promoters of DACT2 and SMOC2, and further documented these CEs in a zebrafish enhancer assay. ConclusionsOur approach yields new insight into the regulation, in dynamic chromatin context, of two Wnt-signaling modulatory genes during synovial joint induction.

developmental biology↗

bioRxiv2022

Rapid specification of human pluripotent stem cells to functional astrocytes

Astrocytes are essential for the formation and maintenance of neural networks through metabolic support, facilitation of synaptic function, and optimization of electrophysiological activity. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Here we present a novel method to efficiently differentiate human pluripotent stem cell (hPSC)-derived neural progenitors to functional astrocytes in 28 days using a culture medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). This approach yields highly pure populations of astrocytes expressing canonical astrocyte markers, which we confirmed by immunofluorescence, flow cytometry and RNA sequencing. Human PSC-derived astrocytes efficiently buffer glutamate and robustly support neural network activity. Co-cultures of hPSC-derived astrocytes and neurons on multi-electrode arrays generated robust network activity within 2 days and synchronous network bursts after 6 days. Whole cell patch-clamp recordings revealed an increased frequency of postsynaptic currents in human hPSC-derived neurons co-cultured with hPSC-derived versus primary rodent astrocytes, consistent with a corresponding increase in synapse density. Furthermore, hPSC-derived astrocytes retained their hominid morphology when transplanted into a mouse brain. In conclusion, we present a novel protocol to obtain functional astrocytes from human pluripotent stem cells, providing a platform for investigating human astrocyte function and neuronal-glial interactions.

neuroscience↗

bioRxiv2021

NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The pro-apoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models we identified an inhibitor of the transcription factor heterodimer CBF{beta}/RUNX1. By genetic gain and loss of function experiments we validated that the mode of action depends on RUNX1 and NOXA. Of note, RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a novel way to target a therapy resistant PDAC, an unmet clinical need. SignificanceRecent evidence demonstrated the existence of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), which resist all current therapies. The paucity of therapeutic options, including a complete lack of targeted therapies, underscore the urgent and unmet medical need for the identification of targets and novel treatment strategies for PDAC. Our study unravels a function of the transcription factor RUNX1 in apoptosis regulation in PDAC. We show that pharmacological RUNX1 inhibition in PDAC is feasible and leads to NOXA-dependent apoptosis. The development of targeted therapies that influence the transcriptional landscape of PDAC might have great benefits for patients who are resistant to conventional therapies. RUNX1 Inhibition as a new therapeutic intervention offers an attractive strategy for future therapies.

cancer biology↗

bioRxiv2021

Zeb2 DNA-binding sites in ES cell derived neuroprogenitor cells reveal autoregulation and align with neurodevelopmental knockout mouse and disease phenotypes.

Perturbation and mechanistic studies have shown that the DNA-binding transcription factor Zeb2 controls cell fate decision and differentiation and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs) Zeb2s strong upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce epitope-tagged Zeb2 from one of its two endogenous alleles. Using ChIP-sequencing, we mapped 2,432 DNA-binding sites of Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major site maps promoter-proximal to Zeb2 itself, and its homozygous removal demonstrates that Zeb2 autoregulation is necessary to elicit proper Zeb2-dependent effects in NPC differentiation. We then cross-referenced all Zeb2 DNA-binding sites with transcriptome data from Zeb2 perturbations in ESCs, ventral forebrain in mouse embryos, and adult neurogenesis from the mouse forebrain V-SVZ. While the characteristics of these neurodevelopmental systems differ, we still find interesting overlaps. This contributes to explaining neurodevelopmental disorders caused by ZEB2 deficiency, including Mowat-Wilson Syndrome.

cell biology↗

bioRxiv2020

SPEN is Required for Xist Upregulation during Initiation of X Chromosome Inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.

developmental biology↗

bioRxiv2019

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform specific start-loss mutations of essential genes can cause genetic diseases

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early onset, therapy resistant seizures and developmental delay. Here we report on 12 individuals from 10 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly and visual disturbance. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A>G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in brain cell types, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modelled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE. Importantly, it also shows that isoform specific start-loss mutations causing expression loss of a tissue relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

genetics↗

bioRxiv2019

Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A and RAD21, cohesin contains in somatic cells one of two orthologous STAG subunits, SA1 or SA2. How these variable subunits affect the function of the cohesin complex is still unclear. SA1- and SA2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of SA1 and SA2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either SA1 or SA2. Following rapid depletion of either subunit, we perform high resolution Hi-C, RNA-sequencing and sequential ChIP studies to show that SA1 and SA2 do not co-occupy individual binding sites and have distinct ways how they affect looping and gene expression. These findings are supported at the single cell level by single-molecule localizations via dSTORM super-resolution imaging. Since somatic and congenital mutations of the SA subunits are associated with cancer (SA2) and intellectual disability syndromes with congenital abnormalities (SA1 and SA2), we verified SA1-/SA2-dependencies using human neural stem cells, hence highlighting their importance for understanding particular disease contexts.

cell biology↗

bioRxiv2019

Models for infantile hypertrophic pyloric stenosis development in patients with esophageal atresia

Patients born with esophageal atresia (EA) have a 30 times higher prevalence of infantile hypertrophic pyloric stenosis (IHPS). This makes sense from a developmental perspective as both the esophagus and the pyloric sphincter are foregut derived structures. EA and IHPS are variable features in several (monogenetic) syndromes. This, and twin and familial studies, indicates a genetic component for both conditions as single entities. We hypothesized that genetic defects, disturbing foregut morphogenesis, are responsible for this combination of malformations. Non-genetic factors could also contribute, as mice exposed to Adriamycin develop EA and in utero diethylstilbestrol exposure is associated with EA.\n\nWe investigated the copy number profiles and protein coding variants of 15 patients with both EA and IHPS. As all parents were unaffected, we first considered dominant (de novo) or recessive inheritance models but could not identify putatively deleterious mutations or recessive variants. We did identify inherited variants in genes either known to be involved in EA or IHPS or important in foregut morphogenesis in all patients. Unfortunately, variant burden analysis did not show a significant difference with unaffected controls. However, the IHPS associated risk SNP rs1933683 had a significantly higher incidence (OR 3.29, p=0.009).\n\nAlthough the genetic variation in likely candidate genes as well as the predisposing locus near BARX1 (rs1933683) suggest a genetic component, it does not fully explain the abnormalities seen in these patients. Therefore, we hypothesize that a combination of high impact genetic, mechanical and environmental factors together can shift the balance to abnormal development.\n\nSummary statementInstead of one affected gene, the higher incidence of IHPS in EA patients is more likely the result of multiple (epi)genetic and environmental factors together shifting the balance to disease development.

genetics↗

bioRxiv2018

MAU2 and NIPBL variants in Cornelia de Lange syndrome reveal MAU2-independent loading of cohesin and uncover a protective mechanism against early truncating mutations in NIPBL

Cornelia de Lange syndrome (CdLS) is a rare developmental disorder caused by mutations in genes related to the cohesin complex. For its association with chromatin, cohesin depends on a heterodimer formed by NIPBL and MAU2, which interact via their respective N-termini. Variants in NIPBL are the main cause of CdLS and result in NIPBL haploinsufficiency.\n\nUsing CRISPR, we generated cells homozygous for an out-of-frame duplication in NIPBL. Remarkably, alternative translation initiation rescued NIPBL expression in these cells and produced an N-terminally truncated NIPBL that lacks MAU2-interaction domain, causing a dramatic reduction of MAU2 protein levels. Strikingly, this protective mechanism allows nearly normal amounts of cohesin to be loaded onto chromatin in a manner that is independent of functional NIPBL/MAU2 complexes and therefore in contrast to previous findings.\n\nWe also report the first pathogenic variant in MAU2, a deletion of seven amino acids important for wrapping the N-terminus of NIPBL within MAU2. The mutation causes dramatic reduction of MAU2 heterodimerization with NIPBL, hence undermining the stability of both proteins.\n\nOur data confirm NIPBL haploinsufficiency as the major pathogenic mechanism of CdLS and give new insights into the molecular mechanisms responsible for this neurodevelopmental disorder. Our work also unveils an alternative translation-based mechanism that protects cells from out-of-frame variants of NIPBL and that may be of relevance in other genetic conditions.

molecular biology↗