Chromatin Nanodomains observed by superresolution microscopy during human neuronal differentiation from induced pluripotent stem cells

CARDOZO GIZZI, ANDRES MAURICIO; REMEDI, MONICA; GASTALDI, LAURA; CACERES, ALFREDO

Rosario

Congreso; Edición Nro. 59 del Congreso Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2023

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

During the course of differentiation, neural progenitors must turn on and off a different set of genes to accomplish a change in cellular phenotype. This is believe to occur thanks to changes in the epigenomic landscape of gene promoters and cis regulatory elements. Moreover, there is a functional relationship between the epigenetic modifications on chromatin and its 3D nuclear organization. Here, we used a human model of neural development derived from induced pluripotent stem cells to study how chromatin post-translational modifications change during the course of differentiation. In particular, we investigated the histone marks H3K27me3 (associated to the Polycomb repressive complex), H3K9me3 (constitutive heterochromatin) and H3K4me3 (associated to active promoters). A non-supervised multiparametric analysis, UMAP, was conducted on microscopy images to separate the different populations in the cell culture. It was found that human neurons are characterized by an intense H3K27me3 mark whereas neural progenitors (Sox2 positive) have relative low levels of this post-translational histone modification. Remarkably, Ezh2, the most active enzyme responsible of the covalent deposition of the mark, is markedly down-regulated as cells progresses to neurons. This lead us to question the role of Ezh2 in neurons. By using immunofluorescence combined with superresolution microscopy we were able to see in single-cells at the nanoscale the domain organization of H3K27me3 (associated to the Polycomb repressive complex) and H3K4me3 (associated to active promoters). Both by expansion and STED microscopy, it was found the existence of nanodomains of H3K27me3 whose size and intensity distribution changed according to cell type.Overall, we found that epigenetic marks intensity and spatial pattern are highly dependent on cell type. By interfering with epigenetic remodeling, i.e. chemical inhibition of activity or knockdown of Ezh1/2 we expect to reveal changes in the expression of key genes in single cells. This initial characterization of the human model lays the ground to study how chromatin spatial organization is required as an additional layer of gene expression regulation. We propose a novel approach to discern new mechanisms of transcriptional regulation in the context of human neuronal differentiation.