MSL2 ensures biallelic gene expression in mammals.

In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter-enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.