Biology subjects

Phiel, C. J.

Publications and source records attributed to Phiel, C. J..

4 recordsLinked to original sources

bioRxiv2024

Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation. Furthermore, we show that LLPS of CBX2 is required for the demarcation and deposition of H3K27me3 and is essential for cellular differentiation. Our findings uncover new functional roles of LLPS in the formation of facultative heterochromatin and unravel a new mechanism by which low-abundant proteins nucleate many other proteins to form compartments that enable them to execute their functions.

molecular biology↗

bioRxiv2023

Mouse Embryonic Stem Cell Pluripotency Factors Regulate RNA Methylation

The pluripotency of embryonic stem cells (ESCs) is actively promoted by a diverse set of factors, including leukemia inhibitory factor (LIF), glycogen synthase kinase-3 (Gsk-3) and mitogen-activated protein kinase kinase (MEK) inhibitors, ascorbic acid, and -ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m6A), which has also been shown to play a role in ESC pluripotency. Therefore, we explored the possibility that these factors converge on this biochemical pathway to promote the retention of ESC pluripotency. Mouse ESCs were treated with various combinations of small molecules, and the relative levels of m6A RNA were measured, as well as the expression of genes marking naive and primed ESCs. The most surprising result was the discovery that replacing glucose with high levels of fructose pushed ESCs to a more naive state and reduced m6A RNA abundance. Our results suggest a correlation between molecules previously shown to promote the retention of ESC pluripotency and m6A RNA levels, strengthening a molecular connection between reduced m6A RNA and the pluripotent state, and provides a foundation for future mechanistic studies on the role of m6A and ESC pluripotency.

molecular biology↗

bioRxiv2023

Vitamin C and Transferrin Reduce RNA Methylation in Mouse Embryonic Stem Cells

Methylation of mRNA on adenosine bases (referred to as m6A) is the most common internal modification of mRNA in eukaryotic cells. Recent work has revealed a detailed view of the biological significance of m6A-modified mRNA, with a role in mRNA splicing, control of mRNA stability, and mRNA translation efficiency. Importantly, m6A is a reversible modification, and the primary enzymes responsible for methylating (Mettl3/Mettl14) and demethylating RNA (FTO/Alkbh5) have been identified. Given this reversibility, we are interested in understanding how m6A addition/removal is regulated. Recently, we identified glycogen synthase kinase-3 (Gsk-3) activity as a mediator of m6A regulation via controlling the levels of the FTO demethylase in mouse embryonic stem cells (ESCs), with Gsk-3 inhibitors and Gsk-3 knockout both leading to increased FTO protein and decreased m6A mRNA levels. To our knowledge, this remains one of the only mechanisms identified for the regulation of m6A modifications in ESCs. Several small molecules that have been shown to promote the retention of pluripotency of ESCs, and interestingly, many have connections to the regulation of FTO and m6A. Here we show that the combination of Vitamin C and transferrin potently reduces levels of m6A and promotes retention of pluripotency in mouse ESCs. Combining Vitamin C and transferrin should prove to be valuable in growing and maintaining pluripotent mouse ESCs.

molecular biology↗

bioRxiv2018

Starvation-dependent regulation of eIF6 through multisite phosphorylation by GSK3β

Eukaryotic translation initiation factor 6 is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. Our studies have uncovered a novel mode of eIF6 regulation by GSK3 that is predominantly active in response to serum starvation. Human eIF6 is phosphorylated by GSK3{beta} at a multisite motif in the C-terminus. Sequential phosphorylation by GSK3{beta} requires phosphorylation at a priming site. In response to serum starvation, eIF6 accumulates in the cytoplasm and this altered localization is dependent on GSK3. Disruption of eIF6 phosphorylation enhances translation inhibition and markedly sensitizes the cells to serum starvation. These results suggest that eIF6 regulation by GSK3{beta} contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.

molecular biology↗