Biology subjects

Gribnau, J.

Publications and source records attributed to Gribnau, J..

At least 19 recordsLinked to original sources

bioRxiv2026

scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells

Single-cell lineage tracing, which often involves CRISPR-Cas-based barcode insertion followed by novel barcode creation, links a living cell's transcriptome to its clonal history. It is often less suitable for short cell differentiation protocols: the barcoding time window does not always align well with that of differentiation, barcode dropout rates can be too high, library preparation remains laborious, and many studies lack appropriate lineage-specific quality control. We developed scRIPT (single-cell Rapid Integrated Platform for Tracing), which combines rapid ribonucleoprotein (RNP)-based barcoding with fluorescent reporter coding sequence insertion, reporter-based enrichment of informative cells by cell sorting, streamlined library preparation, and dual short-read sequencing, coupled to dedicated quality-control metrics for lineage data. scRIPT achieves a low cell dropout rate (<5% of cells presenting without a detectable lineage barcode) and a reliable assignment of barcodes (of >90% of cells). We applied scRIPT to document mouse embryonic stem cell (ESC) differentiation into early hematoendothelial cells and revealed clonal relationships and fate coupling among the emerging cell (sub)types. A subset of cell clones was biased towards single fates, and bifurcating progenitors already express mRNA of canonical regulators of their eventual fate. The results also suggest a heritable, fate-independent clonal memory enriched with imprinted genes. Taken together, scRIPT provides a convenient, rapid, reliable, high-coverage, and quality-controlled platform that combines cell lineage and transcriptome readouts.

developmental biology↗

bioRxiv2026

Joint recording of past and present transcriptomes reconstructs single-cell trajectories

Tracing molecular cell state transitions through development, homeostasis and disease is still beyond reach at single-cell resolution. We present scReMeMber-Seq, a methylation-based cell-memory recorder connecting past transcriptional states with present transcriptome and DNA methylome in individual cells. scReMeMber-Seq leverages pulse induction of DCM-Polr2b (TimeMachine) and single-cell methylome-transcriptome (scMT) sequencing for endpoint readout. In mouse neural stem cells (NSCs) and pancreatic organoids, single-cell DCM methylation profiles (scDCMome) accurately discriminate cell types. Over time, scDCMome also record NSC quiescence-activation state changes across multiple cell divisions. In zebrafish embryos, scDCMome resolve individual transcriptional trajectories from progenitor to neuron. Together, scReMeMber-Seq enables real time reconstruction of cellular trajectories, connecting past and present molecular states in single cells, at genome scale, across experimental systems, in vitro and in vivo.

neuroscience↗

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

NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

Bone marrow fibrosis in myeloproliferative neoplasms arises from interactions between mutant hematopoietic clones and fibrosis-driving stromal cells. We identify Spp1 macrophages as central "communication hubs" integrating inflammatory and fibrotic programs via spatial proximity, ECM signaling, and cytokine activation. Using dual lineage-tracing, single-cell and multiplet RNA-sequencing, and a novel computational method for cell-colocalization and communication analysis named NicheSphere, we show that Spp1 macrophages form core communication hubs with osteoCAR cells, fibroblasts, and megakaryocytes. NicheSphere uncovered two distinct niches: macrophage-enriched compartments driving WNT, JAK-STAT, and TNF cytokine signaling, and a fibrosis-interacting core enriched in TGF-{beta} and ECM glycoproteins. Genetic ablation revealed cooperative roles of stromal and hematopoietic Spp1 in sustaining fibrosis and inflammation. Mechanistically, SPP1 promoted integrin-mediated adhesion, IL-1{beta} secretion, and stromal activation, while IL-1 cytokines induced Spp1 and collagen expression. Loss of Spp1 in hematopoietic progenitors reduced inflammation and restored macrophage function, establishing SPP1 macrophages as therapeutic targets in progressive bone marrow fibrosis.

cancer 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↗

bioRxiv2025

HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation.

The evolution of mammalian sex chromosomes has driven the emergence of mechanisms that balance X-linked gene dosage between male (XY) and female (XX) cells. In females, dosage compensation is achieved through X chromosome inactivation (XCI), initiated by upregulation of the long non-coding RNA Xist, which spreads in cis, recruiting chromatin modifiers to silence gene expression on one X chromosome. Here, we conducted a forward genetic screen and identified X-encoded Host Cell Factor 1 (HCFC1), a member of the COMPASS H3K4 methyltransferase complex, as a dose-dependent XCI-activator. HCFC1 loss results in genome-wide reduction of H3K4me3 at specific regulatory elements and downregulation of nearby genes, including Xist. We show that HCFC1 and YY1 are co-recruited genome-wide to gene regulatory elements. Mass spectrometry analysis confirmed an interaction of HCFC1 and YY1 and uncovered the Integrator complex as another prominent YY1 partner. YY1 depletion results in genome-wide loss of Integrator recruitment at gene regulatory elements and reduced expression of nearby genes, including Xist cis-regulatory genes Jpx and Ftx. These results highlight a co-regulatory role for HCFC1 in COMPASS recruitment and Xist activation, alongside YY1-mediated recruitment of Integrator to Xist regulatory elements and genes to activate female-exclusive XCI.

molecular biology↗

bioRxiv2025

MeCP2 binding and genome-lamina reorganization precede long gene activation during mouse corticogenesis

During corticogenesis, neural gene expression is tightly coordinated by chromatin and epigenetic changes, whose misregulation can lead to neurodevelopmental disorders1-4. The role of spatial genome organization--particularly interactions with the nuclear lamina--during these developmental programs remains poorly understood. Here, we combined in utero electroporation with scDam&T-seq to jointly profile genome-lamina contacts and transcriptomes in single cells of the mouse embryonic cortex. Interestingly, we find extensive genome-lamina reorganization during corticogenesis that is strongly biased towards long neuronal genes ([≥]100 kb), which are associated with neurodevelopmental disorders including autism spectrum disorder. Detachment of these genes frequently precedes transcriptional activation, positioning lamina disengagement as an early gene regulatory event. We identify the methyl CpG binding protein 2 (MeCP2)--mutated in Rett syndrome--as a candidate mediator of this process. MeCP2 binds lamina-associated, hydroxymethylated long genes before their repositioning, suggesting that MeCP2 may play a role in genome-lamina reorganization. These findings suggest a link between prevalent genome-lamina reorganization and MeCP2 regulation to ensure proper spatiotemporal activation of long neuronal genes during corticogenesis.

molecular biology↗

bioRxiv2025

X-chromosome upregulation operates on a gene-by-gene basis at RNA and protein levels

Gene dosage compensation mechanisms are crucial for mammalian development. In mice, recent findings show that cells can sense the number of X chromosomes. Loss or inactivation of one of the two X chromosomes is compensated by upregulating the remaining active X chromosome, a process termed X-chromosome upregulation (XCU). However, how cells sense X-chromosome dosage and induce XCU remains unclear. Here, we show that heterozygous X chromosome fragment deletions in mouse pluripotent stem cells induces XCU in trans, and that compensation takes place at the mRNA and protein level. Furthermore, we found that inducing gene silencing in cis on autosomes induces gene dosage compensation in trans. This work provides significant insights into the molecular foundations of mammalian gene dosage compensation.

developmental biology↗

bioRxiv2025

Single-Cell Roadmap of Early Hemato-Endothelial Development: Functions of Atf3, Zfp711 and Bcl6b

Hematopoiesis is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) that are regulated by transcription factors (TFs) and signaling proteins. We investigated the functions of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b). These TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing (scRNA-seq) and cell identity analysis using Flow Cytometric Analysis (FCA) with TF knockouts (KO) in in-vitro differentiating mouse embryonic stem cells (mESCs) to dissect the function of these TFs in lineage induction, specification, and separation. The Atf3-KO showed an increase of distinct mesodermal subpopulations, but a decrease of endothelial and erythro-myeloid progenitors (EMPs) by downregulation of important genes (i.e. Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, and Zf608) that likely explains the effects on EMPs and the increased expression of interferon-related genes. In Zfp711-KO cells, the number of blood progenitor cells and erythroid cells increased, while the number of endothelial cells decreased. Furthermore, Hoxa expressing mesoderm decreased, while Hoxb expressing mesoderm increased. In contrast, the Bcl6b-KO had no observable effects on early hematopoiesis. In conclusion, we report the function of these three TFs function at different stages of hemato-endothelial lineage specification. HIGHLIGHTSO_LICombined scRNA-seq and FCA on KO mESCs in vitro differentiation enabled unbiased identification of the respective TF functions during specific stages of hematoendothelial lineage specification. C_LIO_LIAtf3-KO: Increased abundance of mesodermal lineages and decreased endothelial lineages and EMPs. - Downregulation of key TF-encoding genes (e.g., Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, Zf608) explain the effects on EMPs. - Generation of a distinct mesodermal subpopulation with high expression of sets of interferon and antiviral-related genes. - Upregulation of interferon and anti-viral-related genes, indicating Atf3 acts as a negative regulator of these genes. C_LIO_LIZfp711-KO: Increased numbers of blood progenitor and erythroid cells. Decreased numbers of endothelial cells. - Shift in mesodermal populations: Hoxa expressing mesoderm decreased, and Hoxb expressing mesoderm increased. - Hoxa9, Hoxa10, Hoxa13, Jun, Jund and Atf3 amongst the downregulated genes. The Atf3 promoter has a potential Zfp711-binding site. - Increased expression in a given Endothelium subtype in endocardium in vivo and in vitro. C_LIO_LIBcl6b-KO: No observable impact on early hematopoiesis. C_LI

developmental biology↗

bioRxiv2025

Wnt-dependent spatiotemporal reprogramming ofbone marrow niches drives fibrosis

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis-inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12 abundant reticular (CAR) progenitor cells resulting in loss of their hematopoiesis-support and upregulation of osteogenic and pro-apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury-specific, Wnt-dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal b-catenin expression is linked to fibrosis in patients and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

molecular biology↗

bioRxiv2025

From Background to Signal: Simultaneously derived Copy Numbers from Methylation-Dependent Sequencing cell-free DNA

Cell-free DNA (cfDNA) analysis offers a powerful, minimally invasive approach to improve cancer care by measuring tumor-specific genomic and epigenetic alterations. Here, we demonstrate the versatility of MeD-seq, a methylation-dependent sequencing assay, for comprehensive cfDNA analysis, including DNA methylation profiling, chromosomal copy number (CN) alterations, and tumor fraction (TF) estimation. MeD-seq-derived CN profiles and TF estimates from 38 colorectal cancer with liver metastases (CRLM) and 5 ovarian cancer patients were highly comparable to shallow whole-genome sequencing (sWGS) validating our approach. For 120 CRLM patients we used MeD-seq CN and TF information in an improved Differential Methylation Model which detected additional significantly Differentially Methylated Regions (DMRs) correlating with TF estimates. Using the identified DMR sets we were subsequently able to distinguish healthy blood donors from CRLM patients with low amounts of circulating tumor DNA (ctDNA) as well. These findings show MeD-seq as an affordable platform for detecting cancer-specific signals directly from plasma without prior tissue-based information. Future work could expand its application to other cancer types, solidifying MeD-seq as a versatile tool for cfDNA profiling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/633371v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@bd440eorg.highwire.dtl.DTLVardef@1befbb7org.highwire.dtl.DTLVardef@17fe9d2org.highwire.dtl.DTLVardef@d0101a_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract of MeD-seq and Copy Number Informed Differential Methylation Analysis Liquid biopsy samples are collected, followed by cfDNA extraction, methylation-dependent enzymatic digestion, Whole Genome Sequencing (WGS) and in silico read selection. Background reads (lacking the recognition site of the methylation-dependent restriction ezyme) are utilized for CN profiling and TF estimation. Methylated reads (with the recognition site of the methylation-dependent restriction enzyme) are counted in CpG islands and integrated with CN information to perform Differential Methylation profiling, enabling a comprehensive assessment of methylation in cfDNA. C_FIG

bioinformatics↗

bioRxiv2025

Characterization of cytotrophoblast cell population dynamics throughout pregnancy

To understand cytotrophoblast (CTB) dynamics during pregnancy, we constructed a single-cell RNA sequencing dataset profiling over 35,000 CTBs isolated from human placental tissues at various gestational stages. Our analysis revealed a robust set of transcripts defining distinct CTB subsets and their spatial relationships within the placenta. We identified a transit-amplifying primitive trophoblast population that may contribute to the CTB pool and found that CTBs located at the smooth chorion form a leak-tight epithelial layer that adapts to low oxygen environments without undergoing syncytialization. Additionally, our study suggests that CTB fate in the villous chorion may be influenced by interactions with the basal lamina, as indicated by a population of BCAM+ CTBs. This subset, isolated from term placental tissue, displayed progenitor capabilities in organoid cultures, supporting the hypothesis that BCAM+ CTBs maintain a progenitor role late in pregnancy.

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

Decoding primitive endoderm - epiblast interactions using mouse ICM embryoids

Stem cell-based embryo models are promising alternatives for investigating early embryogenesis. We introduce two distinct models to replicate the dynamics between extra-embryonic endoderm and epiblast during mouse embryonic development. Inducible Gata6 (iGata6) embryoid bodies (EBs), exclusively derived from iGata6 embryonic stem (ES) cells, are valuable for modeling the position-dependent development of the primitive endoderm. Inner cell mass (ICM) embryoids, conversely, efficiently formed by aggregating wild-type and iGata6 ES cells, accurately and at a comparable pace simulate in vivo development from E3.5 to E7.5. Notably, ICM embryoids model cell sorting and through a rosette-like stage, the transition of the epiblast from naive to primed pluripotency. Furthermore, the absence of extra-embryonic ectoderm-like cells in this model, directs both the epiblast and visceral endoderm towards an anterior developmental fate. As such, iGata6 EBs and ICM embryoids are powerful tools for advancing our understanding of cell fate decisions during early embryonic development in mice.

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↗