Quantitative imaging of Oct4 and HP1 in embryonic stem cells

GUBERMAN ALEJANDTA; VERNERI PAULA; OSES CAMILA; LEVI VALERIA

Congreso; reunion conjunta de biociencias; 2017

The dynamical interactions of transcription factors (TFs) and DNAtargets play a fundamental role in different stages involved in gene expression.Knowing the transcription machinery distributes among different sub-nucleardomains, we seek to understand how the architecture of the nucleus modulatesthe transcriptional response.Embryonic stem cells (ESC) have an unlimited possibility of self-renewal andare pluripotent, that means that they can give rise to all adult cell types. Theseproperties make ESC studies relevant for understanding embryo development.Pluripotency depends on specific TFs such as Oct4, Sox2 and Nanog, whichinduce genes necessary to preserve an undifferentiated state and repressgenes related to differentiation.In this work, we used fluorescence correlation spectroscopy (FCS) analyses toquantitatively explore the dynamical organization of TFs in the nucleus of ESC.With this aim, we transfected the cells with vectors encoding TFs or chromatin-associated proteins fused to enhanced green fluorescent protein (GFP), andmeasured fluorescence fluctuations as a function of time using confocalmicroscopy. We focused our studies on the dynamics of Oct4 in undifferentiatedESC and analyzed the FCS data with a model that considers fast and slowinteractions with DNA targets. We verified that Oct4 partitions between fast andslow sites in undifferentiated ESC and that this equilibrium changes when ESCare subjected to a differentiation protocol. Moreover, the knock-out of achromatin remodeler (KAT6B -/-) also modulates the dynamical distribution ofthe transcription factor and of the heterocromatin-associated protein HP1,showing differences respect to the wild type ESC line.These results show the fine modulation of TF-DNA interactions in the cellnucleus and thus these studies may provide important clues for understandingthe transcriptional response, especially at a not so well studied system such asESC. These approaches could help us to understand early embryodevelopment in future studies.