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

CARDOZO-GIZZI A; REMEDI MM; GASTALDI L; CACERES A

Rosario

Congreso; Congreso SAIB 2023. Reunion Anual, Edicion LIX; 2023

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular

During the course of differentiation, neural progenitors must turn on and off a different set ofgenes to accomplish a change in cellular phenotype. This is believe to occur thanks tochanges in the epigenomic landscape of gene promoters and cis regulatory elements.Moreover, there is a functional relationship between the epigenetic modifications onchromatin and its 3D nuclear organization.Here, we used a human model of neural development derived from induced pluripotent stemcells to study how chromatin post-translational modifications change during the course ofdifferentiation. In particular, we investigated the histone marks H3K27me3 (associated to thePolycomb repressive complex), H3K9me3 (constitutive heterochromatin) and H3K4me3(associated to active promoters). A non-supervised multiparametric analysis, UMAP, wasconducted on microscopy images to separate the different populations in the cell culture. Itwas found that human neurons are characterized by an intense H3K27me3 mark whereasneural progenitors (Sox2 positive) have relative low levels of this post-translational histonemodification. Remarkably, Ezh2, the most active enzyme responsible of the covalentdeposition of the mark, is markedly down-regulated as cells progresses to neurons. Thislead us to question the role of Ezh2 in neurons.By using immunofluorescence combined with superresolution microscopy we were able tosee in single-cells at the nanoscale the domain organization of H3K27me3 (associated to thePolycomb repressive complex) and H3K4me3 (associated to active promoters). Both byexpansion and STED microscopy, it was found the existence of nanodomains of H3K27me3whose size and intensity distribution changed according to cell type.Overall, we found that epigenetic marks intensity and spatial pattern are highly dependent oncell type. By interfering with epigenetic remodeling, i.e. chemical inhibition of activity orknockdown of Ezh1/2 we expect to reveal changes in the expression of key genes in singlecells. This initial characterization of the human model lays the ground to study howchromatin spatial organization is required as an additional layer of gene expressionregulation. We propose a novel approach to discern new mechanisms of transcriptionalregulation in the context of human neuronal differentiation.