Sari Pennings’s research while affiliated with University of Edinburgh and other places

Rett syndrome is a severe neurodevelopmental disorder in girls, underpinned by mutations in the X-linked gene MECP2. In their recent work (Frasca et al, 2024), Frasca and colleagues identified a novel pathway involving interferon-gamma (IFNγ) that could pave the way to potential therapies.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in 2i conditions results in redistribution of polycomb (H3K27me3) away from its target loci. Here we demonstrate that 3D genome organisation is also altered in 2i. We found chromatin decompaction at polycomb target loci as well as loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground-state, we are able to restore the H3K27me3 distribution, and polycomb-mediated 3D genome organisation that is characteristic of primed ESCs grown in serum, to ESCs in 2i. However, these cells retain the functional characteristics of 2i ground state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organisation but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

Heart development in mammals is followed by a postnatal decline in cell proliferation and cell renewal from stem cell populations. A better understanding of the developmental changes in cardiac microenvironments occurring during heart maturation will be informative regarding the loss of adult regenerative potential. We reevaluate the adult heart’s mitotic potential and the reported adult cardiac stem cell populations, as these are two topics of ongoing debate. The heart’s early capacity for cell proliferation driven by progenitors and reciprocal signalling is demonstrated throughout development. The mature heart architecture and environment may be more restrictive on niches that can host progenitor cells. The engraftment issues observed in cardiac stem cell therapy trials using exogenous stem cells may indicate a lack of supporting stem cell niches, while tissue injury adds to a hostile microenvironment for transplanted cells. Engraftment may be improved by preconditioning the cultured stem cells and modulating the microenvironment to host these cells. These prospective areas of further research would benefit from a better understanding of cardiac progenitor interactions with their microenvironment throughout development and may lead to enhanced cardiac niche support for stem cell therapy engraftment.

Recent progress in interpreting comprehensive genetic and epigenetic profiles for human cellular states has contributed new insights into the developmental origins of disease, elucidated novel signalling pathways and enhanced drug discovery programs. A similar comprehensive approach to decoding the epigenetic readouts from chemical challenges in vivo would yield new paradigms for monitoring and assessing environmental exposure in model systems and humans.

Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry. Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin. Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.