nuclei-isolation-from-mouse-cardiac-progenitor-cells-for-epigenome

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Aix-Marseille University

OCT4 encoded by pou5f1 is one of the most ancient and early transcription factors identified in the embryo. It has been longwise recognized as a gatekeeper for pluripotency of embryonic stem (ES) cell. Uncovered twenty years ago, its fame was built up from its key role in maintaining embryonic stem cell pluripotency in 1998. Since, OCT4 was reported to also instruct stem cell fate through a gene dosage effect. It reached recently a novel glorious hit with its master role in reprogramming somatic cells.

[The dual role of OCT4].

Short- and long-scales intra- and inter-chromosomal interactions are linked to gene transcription, but the molecular events underlying these structures and how they affect cell fate decision during embryonic development are poorly understood. One of the first embryonic cell fate decisions (that is, mesendoderm determination) is driven by the POU factor OCT4, acting in concert with the high-mobility group genes Sox-2 and Sox-17. Here we report a chromatin-remodelling mechanism and enhancer function that mediate cell fate switching. OCT4 alters the higher-order chromatin structure at both Sox-2 and Sox-17 loci. OCT4 titrates out cohesin and switches the Sox-17 enhancer from a locked (within an inter-chromosomal Sox-2 enhancer/CCCTC-binding factor CTCF/cohesin loop) to an active (within an intra-chromosomal Sox-17 promoter/enhancer/cohesin loop) state. SALL4 concomitantly mobilizes the polycomb complexes at the Soxs loci. Thus, OCT4/SALL4-driven cohesin- and polycombs-mediated changes in higher-order chromatin structure mediate instruction of early cell fate in embryonic cells.

A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage.

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3 pacemaker cells. Here, we have isolated and characterized the Tbx3 cells from  knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3 pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3 sinoatrial nodal cells and Nppb-Katushka atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and , the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the  allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program.

The components of the cardiac conduction system, responsible for coordinated activation of the heart chambers, are well defined and their cells differ in gene expression profile and phenotype from those of the surrounding working myocardium. Yet, when and on what basis the myocardium of each of the conduction system components become distinguishable from other myocardium during heart development has not been well established. To identify and assess cell type-specific expression profiles and differentiation markers, we performed transcriptome analysis on fluorescence activated cell sorted purified conduction system (Venus) and chamber myocardial cells (Katushka) of Tbx3;TgNppb(Katushka) double transgenic mouse fetuses. We found that transcripts associated with nervous system development and ion channel activity were enriched in Tbx3 conduction system cells, whereas transcripts associated with mitochondrial function, muscle contraction and fatty acid metabolism were enriched in the Nppb working myocardium. We analyzed spatio-temporal expression patterns of several candidate markers (Cacna2d2, Cacna1g, Ephb3, Tnni1), reviewed those of established conduction system markers (Tbx3, Hcn4, Gja5, Cntn2), and placed the patterns in the context of conduction system development. The overview indicates that different properties of conduction system components develop gradually and at different developmental stages, and that chamber myocardium gradually differentiates and diverges from conduction system myocardium until after birth.

Article	Year
[The dual role of OCT4]. Médecine sciences : M/S\| PubMed ID: 20412747	2010
A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage. Nature communications\| PubMed ID: 25851587	2015
Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program. Development (Cambridge, England)\| PubMed ID: 30936179	2019
Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 31306714	2020

Sonia Stefanovic

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