engleski

The effect of Drosophila melanogaster embrionic extract on (partial) nuclear reprogramming

It is known that differentiated cells can be reprogrammed when exposed to an oocyte or an embryonic extract. In this work, we followed early reprogramming events in S2 cells treated with DREX (Drosophila early-embryo extract). We have observed that a simultaneous loss of somatic H1 linker histone is not necessary for the chromatin incorporation of the embrionic variant of the linker histone, dBigH1. Upon screening, dBigH1 was present at all inspected loci, and no specifically enriched or depleted regions were detected. We have also shown that dBigH1 incorporation into chromatin is an ATP-facilitated event. Furthermore, heterochromatin reorganization upon incubation in DREX is observed. Heterochromatic foci lose their usual distribution pattern, starting to be more uniformly dispersed along the nuclei, with slight accumulation on the periphery of the nuclei. Following that, in a certain number of nuclei HP1a foci start to re-appear, not in their usual distribution pattern, but extruded from the surface of the nuclei. ChIP-qPCR experiments performed to determine H3K9me2 occupancy at different heterochromatin loci indicate that these epigenetic marks are not being removed during the processes of heterochromatin remodeling. We have also observed that disassembly of lamin occurs upon exposure to embryonic extract, and that it is positively correlating with the disappearance of heterochromatic foci. It has also been confirmed that linker histone incorporation alone is not enough to produce previously described effects. As reprogramming process have shown to be accompanied by epigenetic changes, mostly on pluripotency-related or developmentally regulated genes, we explored 5 genes that show very high expression only in the early embryo. We revealed that all inspected genes show significant increase in H3K4me3 at their promoters upon exposure to DREX. Taking into consideration that at least three events characteristic for pluripotent cells are present in our system (dissapearance of heterochromatic foci, dissasembly of lamin, and changes in epigenetic marks suggesting the activation of early-embryo genes), we can conclude that processes following DREX incubation induce the transition of S2 cells from a differentiated to a (partially) reprogrammed state.

embrionic extract, chromatin, histone variant, dBigH1, nuclear reprogramming

nije evidentirano