Abstract
BackgroundOsmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown.MethodologyUsing hypertonic induction of the aldose reductase (AR) gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s) around the OREs. The loss of nucleosome(s) was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBP-dependent hyperacetylation of histones that spanned the 5′ upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone.SignificanceOur findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled.
Highlights
Exposure of mammalian cells to hypertonic stress induces osmotically obliged water efflux that results in cell shrinkage
As histone modifications regulate the accessibility of chromatin and gene activity [42,43,44], we first examined if hypertonicity induces, in vicinity of the OREs, histone acetylation, which is known to regulate chromatin folding [45] and is a hallmark of gene activation [46,47,48]
We carried out chromatin immunoprecipitation (ChIP) followed by quantitative PCR amplification of a 160 bp amplicon that spanned the OREs (Fig. 1A, ARORE)
Summary
Exposure of mammalian cells to hypertonic stress induces osmotically obliged water efflux that results in cell shrinkage. Chronic increase in intracellular ionic strength may inhibit protein synthesis and induce growth arrest [2]. Such deleterious conditions are counteracted by the gradual replacement of electrolytes with organic osmolytes including sorbitol, betaine, myo-inositol, taurine and glycerophosphocholine. These osmolytes replace intracellular electrolytes and preserve cell volume and osmolality without perturbing macromolecular structure and function [3,4]. Osmotic Response Element-Binding Protein (OREBP), known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. How OREBP interacts with target DNA, known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown
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