Biology subjects

Tan, B. F.

Publications and source records attributed to Tan, B. F..

4 recordsLinked to original sources

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

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↗

bioRxiv2023

Specific genomic targeting of the RNF12/RLIM E3 ubiquitin ligase selectively programmes developmental transcription

The E3 ubiquitin ligase RNF12/RLIM controls developmental gene expression and is mutated in the X-linked intellectual disability disorder Tonne-Kalscheuer syndrome (TOKAS). However, the mechanisms by which RNF12 E3 ubiquitin ligase activity controls specific gene expression signatures are not known. Here, we show that chromatin forms a regulatory platform for RNF12 substrate ubiquitylation and transcriptional patterning. RNF12 is recruited to specific genomic regions via a distinct consensus sequence motif, which enables targeting to key transcription factor substrate REX1. Mechanistically, RNF12 chromatin recruitment is largely REX1 independent, but is achieved via the conserved basic region (BR) adjacent to the RING domain. This region is critical for REX1 ubiquitylation on chromatin and downstream RNF12-dependent gene regulation. Furthermore, we find that RNF12 N-terminal sequences suppress chromatin recruitment and substrate ubiquitylation, uncovering a previously unappreciated autoinhibitory mechanism that governs genome targeting. Taken together, our results provide insight into mechanisms by which selective substrate targeting of an E3 ubiquitin ligase enables specific programming of gene expression.

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↗