CONICET | Buscador de Institutos y Recursos Humanos

Pluripotency ofembryonic stem cells (ESC) depends on transcription factors (TFs) such as Oct4,Sox2 and Nanog, which induce genes necessary to preserve an undifferentiatedstate and repress genes related to differentiation. It has beenproposed that gene expression not only depends on the expression levels of TFsbut also on dynamic changes in intracellular distribution that could affectTF-chromatin interactions. The dynamicalinteractions of TFs and DNA targets play a fundamental role indifferent stages involved in gene expression, highly relevant in the control ofcell differentiation and development. Therefore, exploringthe transcription machinery?s dynamics in living cells is essential tounderstand how the dynamical organization of nucleus and specifically, thedynamics of chromatin-associated proteins and TFs influence the transcriptionaloutput. In this work,we used anon-invasive microscopy method denominated fluorescence correlationspectroscopy (FCS) to quantitatively explore the dynamical organization of TFsin the nucleus of ESC. With this aim, we used stable ESC lines encoding Oct4 orSox2 C-terminally fused to a yellow fluorescent protein. We focused our studieson the dynamics of Oct4 and Sox2 in undifferentiated and differentiated ESC.Besides, we transfected the cells with vectors encoding chromatin-associatedproteins fused to mCherry and analyzed how chromatin remodel duringdifferentiation. Our results show that in the undifferentiated state,Oct4 is distributed homogenously in the nucleus and analyzed FCS data fit witha model that considers fast and slow interactions with chromatin targets. Inaddition, when cells are submitted to a differentiation protocol, the processis accompanied by a repartitioning of Oct4 and Sox2 in distinguishable foci. Wealso found that this change in TFs dynamics is accompanied with changes in TFs-chromatininteraction times and chromatin remodeling.These studies may provideimportant clues for understanding how thefine modulation of TF-DNA interactions in the cell nucleus regulates the transcriptional response.These approaches could help us to understand early embryo development in futurestudies