Dynamics of Oct4, Nanog and HP1 in embryonic stem cells

LEVI V; COSENTINO S; OSES C; GUBERMAN A

Taller; IV Taller de Biología Celular y del Desarrollo; 2018

Transcription factors (TFs) dynamic interactionswith DNA targets depend, among multiple parameters, on chromatin landscape.Despite TFs-chromatin interactions play a relevant role in the modulation of geneexpression we still do not know how the complex temporal and spatial network ofinteractions defines transcriptional response in stem cells (SCs).Embryonic SCs (ESC) studies are relevant for understanding embryo development and for theirpotential clinical applications. These cells have two importantproperties: an unlimited possibility of self-renewaland pluripotency, which depends on specific TFs such as Oct4, Sox2 andNanog that repress genes involved in differentiation and induce genes thatpreserve an undifferentiated state.In this work, we used fluorescence correlation spectroscopy (FCS) analysisto quantitatively explore the dynamical organization of TFs in the nucleus of ESCwild type (wt) and an ESC line knock-out for a chromatin remodeler, the histoneacetyltransferase Kat6B (Kat6B-/-). Since histone acetylation usually makes amore permissive chromatin, we expected a different dynamical distribution ofTFs and heterochromatin associated proteins between Kat6B -/- and wt ESCs. With the aim of comparing these dynamics in ESC wild type with Kat6B-/-,we transfected the cells with vectors encoding Oct4, Nanog or HP1 fused toenhanced green fluorescent protein (GFP), and measured fluorescence fluctuationsas a function of time using confocal microscopy. We analyzed the FCS data witha model that considers fast and slow interactions with DNA targets. Results showed statistical significantdifferences between both cell lines for both TFs and HP1, which are consistentwith a less permissive chromatin in Kat6B -/- ESC line. These results mayprovide important clues for understanding the transcriptional response. Furthermore, they show that FCS is a useful tool to evaluate howbiological processes modulate TFs partition in the nucleus and could help us to understandearly embryo development in future studies.