Biology subjects

Boers, R.

Publications and source records attributed to Boers, R..

8 recordsLinked to original sources

bioRxiv2026

Time-resolved single-cell state tracing exposes bidirectional state dynamics during differentiation

Differentiation dynamics have been challenging to study across time as measuring cell state, transcriptionally or epigenetically, typically requires its destruction. Here, we developed single-cell state tracing using the DCM-Time machine to enable the characterization of two transcriptomes, separated in time in single cells during epithelial cell differentiation in the intestine of living animals, and during neuronal differentiation in culture. We show that retrospective transcriptional histories and current states are consistent with bidirectional movements along differentiation trajectories both in vivo and in vitro that become rarer as cells mature. Furthermore, we observe rare transcriptome conversions, across canonical lineage boundaries. Our data support a model in which differentiating cells occasionally fail to stabilize lineage-associated programs, enabling reversals and subsequent resolution toward alternative lineage states.

developmental biology↗

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↗

bioRxiv2025

XIST Drives X-Chromosome Inactivation and Safeguards Female Extraembryonic Cells in Humans

Dosage compensation of sex chromosomes through X-chromosome inactivation (XCI) is required for mice extra-embryonic tissue growth and embryo development. The species specificity in mechanisms and timing leading to XCI during early embryogenesis, however, left the key question of the interdependence between XCI and human development open. Here, we show that the differentiation of naive human pluripotent stem cells to trophoblast stem cells and extraembryonic mesoderm cells triggers XCI. The inactive X chromosome, however, displays an atypical chromatin state, lacking classical enrichment of heterochromatin markers and DNA methylation. We demonstrate that extraembryonic differentiation and XCI are kinetically and functionally linked. Using loss of function approaches, we prove that XIST is required for human XCI establishment. We also reveal that XCI is key for the survival of human female extraembryonic cells. Our work therefore links XCI to the formation of extraembryonic annexes, with important consequences for human reproductive biology. HIGHLIGHTSO_LINaive hPSCs to EXMCs and TSCs differentiation recapitulates human XCI C_LIO_LIThe Xi has an unusual chromatin status in human extraembryonic cells C_LIO_LIXIST is required for the establishment of human XCI C_LIO_LIXCI supports healthy development of human female extraembryonic cells C_LI

developmental biology↗

bioRxiv2025

Human DCM-time machine unravels cell state changes during primitive gut tube differentiation

Cell state changes in development and differentiation are directed by gene and enhancer activity changes, the dynamics of which are difficult to study in real time. To be able to define lineage paths and monitor time resolved cell state changes during pluripotent stem cell differentiation, we introduced the DCM-time machine technology (DCM-TM) into human induced pluripotent stem cells (iPSCs). We demonstrate that, at time of induction, human DCM-TM labels active genes and enhancers. In addition, we find that the majority of the DCM methylation labels are propagated during S-phase, which makes it well suited to trace gene and enhancer activity during cell state changes. As proof of concept, we applied the DCM-time machine to study differentiation from iPSCs towards definitive endoderm and primitive gut tube cells. In a comparative analysis with scRNA-seq, we show the capacity of the system to label gene and enhancer activity during differentiation and trace back gene and enhancer activity over time across multiple cell divisions. In addition, we demonstrate that combining DCMTM with methylated DNA sequencing (MeD-seq) enables the detection of CpG methylation changes, which can be correlated with gene and enhancer activity dynamics. Human DCM-TM provides a novel genome-wide lineage tracing tool for iPSCs and will be a significant contribution to the understanding of healthy and pathogenic embryonic development.

developmental biology↗

bioRxiv2024

Genome-wide methylation profiling of cell-free DNA in maternal plasma using Methylated DNA Sequencing (MeD-seq)

BackgroundPlacental-originated cell-free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD-seq) is well-compatible with low cfDNA concentrations and has a high genome-wide coverage. We therefore aim to investigate the feasibility of genome-wide methylation profiling of first trimester maternal cfDNA using MeD-seq, by identifying placental-specific methylation marks in cfDNA. MethodsWe collected cfDNA from non-pregnant controls (female n=6, male n=12) and pregnant women (n=10), first trimester placentas (n=10), and paired preconceptional and first trimester buffy coats (total n=20). Differentially methylated regions (DMRs) were identified between pregnant and non-pregnant women. We investigated placental-specific markers in maternal cfDNA, including RASSF1 promoter and Y-chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation. ResultsWe identified 436 DMRs between cfDNA from pregnant and non-pregnant women which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p<0.0001). Differential methylation of Y-chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats, indicating a placental and immune-cell contribution to the pregnancy-specific cfDNA methylation signature. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental-specific DMRs were more prominent than RASSF1. ConclusionsMeD-seq can detect (novel) genome-wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. This study supports future research into maternal cfDNA methylation using MeD-seq. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/610227v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@b8fd55org.highwire.dtl.DTLVardef@ffe942org.highwire.dtl.DTLVardef@12afbf4org.highwire.dtl.DTLVardef@1036a6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Studies investigating the maternal cell-free DNA (cfDNA) methylome are scarce and generally technologically challenging. We identified 436 autosomal differentially methylated regions (DMRs) between cfDNA from pregnant and non-pregnant women, using the innovative methylated DNA sequencing (MeD-seq) technique. Y-chromosomal methylation could determine fetal sex, we show hypermethylation of the placental-marker RASSF1, and identify 16 novel placental-specific markers in maternal cfDNA including DMRs related to TMEM240, DHRS3, and PCMTD2. This pilot study supports future research into the maternal cfDNA methylome using MeD-seq.

genomics↗

bioRxiv2024

DNA methylation database for gynecological cancer detection, classification and assay development

Changes in the genome wide DNA methylation landscape are hallmarks of cancer cells and precursor lesions of cancers. To capitalize on utilizing DNA methylation for detection and classification of cancer, we generated a DNA methylation database of gynecological cancers and associated healthy tissues using Methylated DNA sequencing (MeD-seq). We show that target cell enrichment to generate the database is crucial for marker discovery and report a wide range of novel biomarkers for classification and tissue of origin determination of gynecological cancers. We developed a subset of these novel biomarkers, both intragenic and intergenic, into a qMSP assays that detect all gynecological cancers at once or specific gynecological cancer subtypes, as well as cancers that are not part of our database. The database generated in this study not only provides the foundation for cancer detection, classification and biomarker discovery, but also for treatment monitoring of cancers using MeD-seq on liquid biopsies.

cancer biology↗

bioRxiv2023

XIST dampens X chromosome activity in a SPEN-dependent manner during early human development

XIST long non-coding RNA is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female pre-implantation embryos without triggering X chromosome silencing. The long non-coding RNA XACT co-accumulates with XIST on active Xs and may antagonize XIST function. Here we used human ES cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during pre-implantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and attenuates transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional prior to XCI, confirms the existence of a transient process of X chromosome dosage compensation, and reveals that X chromosome inactivation and dampening rely on the same set of factors.

molecular biology↗

bioRxiv2021

Retrospective analysis of enhancer activity and transcriptome history

Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. Here, we utilize an inducible DCM-RNA-polymerase-subunit-b fusion protein, to label active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. We applied this DCM-time-machine (DCM-TM) technology to study intestinal homeostasis, following enterocyte differentiation back in time, revealing rapid and simultaneous activation of enhancers and nearby genes during intestinal stem cell (ISC) differentiation. We provide new insights in the absorptive-secretory lineage decision in ISC differentiation, and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes in development and differentiation.

developmental biology↗