Biology subjects

Blotenburg, M.

Publications and source records attributed to Blotenburg, M..

5 recordsLinked to original sources

bioRxiv2024

T-ChIC: multi-omic detection of histone modifications and full-length transcriptomes in the same single cell

Epigenetic mechanisms, including histone modifications, are key regulators of transcription and influence cellular differentiation. To uncover the interplay between histone modifications and transcription, we developed T-ChIC (Transcriptome + Chromatin ImmunoCleavage), which detects full-length transcripts and histone modifications in the same single cell. We applied T-ChIC to gastruloids, an in vitro model containing cell types derived from all three germ layers. During gastruloid development we observed a switch in transcription start site usage from multiple regions marked by H3K4me3 to one marked start site per gene. H3K27me3 coverage transitions from global marking of non-transcribed regions towards localised marking of repressive regions. Upon lineage commitment, bivalent regions are sequentially resolved towards a germ layer-specific state, where endoderm retains a high abundance of H3K27me3 while ectoderm displays a widespread loss. Together, we propose a model for a cell-intrinsic epigenetic timer, which ensures sequential perceptiveness of pluripotent cells to transcriptional activation of germ layer-specific genes.

genomics↗

bioRxiv2024

Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid development

By aggregating low numbers of mouse embryonic stem cells (mESCs) followed by exposure to Wnt activation, structures termed gastruloids are produced that display an anteroposterior organisation of cell types derived from all three germ layers. Since current gastruloid protocols display considerable heterogeneity between experiments in terms of morphology, elongation efficiency, and cell type composition, we investigated whether an optimised mESC pluripotency state would provide more consistent results. By growing mESCs in different intervals of ESLIF and 2i medium the pluripotency state of cells was modulated, and mESC culture as well as the resulting gastruloids were analysed. Microscopy analysis showed a pre-culture-specific effect on gastruloid formation, in terms of efficiency, elongation index and reproducibility. RNA-seq analysis of the mESC start population confirmed that short-term pulses of 2i and ESLIF modulate the pluripotency state, and result in different cellular states. With multiple epigenetic regulators among the top differentially expressed genes we further analysed genome-wide DNA methylation and H3K27me3 distributions. We observed the same divergence between conditions, most dominantly displayed in the promoter regions of developmental regulators. Lastly, when we investigated the cell type composition of gastruloids grown from these different pre-cultures, we observed that mESCs subjected to 2i-ESLIF preceding aggregation generated gastruloids more consistently, including more complex mesodermal contributions as compared to the ESLIF-only control. This indicates that optimisation of the mESCs pluripotency state allows the modulation of cell differentiation during gastruloid formation.

developmental biology↗

bioRxiv2021

Salt Inducible Kinase activation and IRE1-dependent intracellular ATP depletion to form Sec bodies in Drosophila cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation. We find that two pathways are critical. The first is a SIK dependent pathway induced by salt (NaCl) stress in a necessary and sufficient manner. The second is the activation of IRE1 (one of the key kinases mediating the Unfolded Protein Response) by absence of amino- acids, which partly leads to the depletion of intracellular ATP. However, IRE1 activation is not sufficient to induce Sec body formation and needs to be combined to salt stress. This works pioneers the role of SIK in phase transition and re-enforces the role of IRE1 as a metabolic sensor for the level of circulating amino-acids.

cell biology↗

bioRxiv2019

Dppa2/4 target chromatin bivalency enabling multi-lineage commitment

Bivalent chromatin marks developmental promoters in pluripotent cells, yet their targeting and precise impact on lineage commitment remains unclear. We uncover Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as epigenetic priming factors, establishing chromatin bivalency. Single-cell transcriptomics and differentiation assays reveal Dppa2/4 double knockout embryonic stem cells fail to exit pluripotency and differentiate efficiently. Dppa2/4 associate with COMPASS and Polycomb complexes and are required to recruit and maintain their binding at a subset of developmentally important bivalent promoters which are characterised by low expression and poised RNA polymerase. Consequently, upon Dppa2/4 knockout, these dependent promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel targeting principle for bivalency to developmental promoters, poising them for future lineage specific activation.

molecular biology↗

bioRxiv2018

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

The molecular regulation of zygotic genome activation (ZGA) in mammals remains poorly understood. Primed mouse embryonic stem cells contain a rare subset of \"2C-like\" cells that are epigenetically and transcriptionally similar to the two cell embryo and thus represent an ideal system for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed exclusively in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA either in a Dux dependent or independent manner. Here we performed a candidate-based overexpression screen, identifying, amongst others, Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as positive regulators of 2C-like cells and ZGA transcription. In the germ line, promoter DNA demethylation coincides with upregulation of Dppa2 and Dppa4 which remain expressed until E7.5 when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during iPSC reprogramming at the time 2C-like ZGA transcription transiently peaks. Through a combination of overexpression, knockdown, knockout and rescue experiments, together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we tease apart the molecular hierarchy in which the 2C-like transcriptional programme is initiated and stabilised. Dppa2 and Dppa4 require Dux to initiate 2C-like ZGA transcription, suggesting they act upstream by directly regulating Dux. Supporting this, ChIP-seq analysis revealed Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild type cells, but, in contrast to Dux, can no longer do so in Dppa2/4 double knockout cells, suggesting it may act to stabilise rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux upregulation and activation of the 2C-like transcriptional programme which is subsequently reinforced by Zscan4c.

molecular biology↗