QUANTITATIVE IMAGING OF NUCLEAR PROTEINS IN EMBRYONIC STEM CELLS

VÁZQUEZ ECHEGARAY CAMILA; GUBERMAN ALEJANDRA; VERNERI PAULA; LEVI VALERIA

Chascomús

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2018

Pluripotency of embryonic stem cells (ESC) depends on transcription factors (TFs) such as Oct4, Sox2 and Nanog, which induce genes necessary to preserve an undifferentiated state and repress genes related to differentiation. It has been proposed that gene expression not only depends on expression levels of TFs but also on dynamic changes in intracellular distribution that could affect TF-chromatin interactions. The dynamical interactions of TFs and DNA targets play a fundamental role in different stages involved in gene expression, highly relevant in the control of cell differentiation and development. Therefore, exploring the transcription machinery?s dynamics in living cells is essential to understand how the dynamical organization of nucleus and specifically, the dynamics of chromatin-associated proteins and TFs influence the transcriptional output. In this work, we used a non-invasive microscopy method namely fluorescence correlation spectroscopy (FCS) to quantitatively explore the dynamical organization of TFs in the nucleus of ESC. With this aim, we used stable ESC lines encoding Oct4 or Sox2 C-terminally fused to a yellow fluorescent protein. We focused our studies on the dynamics of Oct4 and Sox2 in undifferentiated and differentiated ESC. Besides, we transfected the cells with vectors encoding chromatin-associated proteins fused to mCherry and analyzed how chromatin remodel during differentiation. Our results shows that in an undifferentiated state, Oct4 it?s distributed homogenously in the nucleus and analyzed FCS data fits with a model that considers fast and slow interactions with chromatin targets. In addition, when cells are submitted to a differentiation protocol, the process is accompanied by a repartitioning of Oct4 and Sox2 in distinguishable foci. We also found that this change in TFs dynamics is accompanied with changes in TFs-chromatin interaction times and chromatin remodeling. These studies may provide important clues for understanding how the fine modulation of TF-DNA interactions in the cell nucleus regulates the transcriptional response. These approaches could help us to understand early embryo development in future studies.