To address a direct role of Lsh in chromatin remodeling at an endogenous locus we employed an in vitro ES cell system that allowed for tethering of Lsh to a specific site. The ES cells were genetically modified and carried five Gal4 binding sites at the Oct4 promoter region of the Oct4 gene and a GFP reporter inserted in the first exon to monitor expression. We ectopically expressed Lsh as a fusion protein with the DNA binding domain of the yeast transcription factor Gal4 in ES cells. As control, we expressed the Gal4 DNA binding alone. We detected higher nucleosome density after recruitment of Lsh concomitant with altered histone modifications (reduced H3K4me3 and augmented H3K27me3 and H3K9me3). Upon differentiation of ES cells tethered Lsh induced repression of the GFP reporter construct associated with a further increase of repressive histone modifications. Together with the fact that Lsh belongs to a class of nucleosome remodelers and that Lsh requires ATP function to alter nucleosome position, our data indicate a direct (localized effect) of Lsh on chromatin accessibility at the endogenous recruitment site. This suggests that Lsh mediated changes of nucleosome density are a primary consequence of Lsh function. Revealing Lsh molecular function helps to define its role in the ICF syndrome in the future.
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