RNA-Seq analysis of high NaCl-induced gene expression

Abstract

High extracellular NaCl is known to change expression of numerous genes, many of which are regulated by the osmoprotective transcription factor nuclear factor of activated T cells-5 (NFAT5). In the present study we employed RNA-Seq to provide a comprehensive, unbiased account of genes regulated by high NaCl in mouse embryonic fibroblast cells (MEFs). To identify genes regulated by NFAT5 we compared wild-type MEFs (WT-MEFs) to MEFs in which mutation of the NFAT5 gene inhibits its transcriptional activity (Null-MEFs). In WT-MEFs adding NaCl to raise osmolality from 300 to 500 mosmol/kg for 24 h increases expression of 167 genes and reduces expression of 412. Raising osmolality through multiple passages (adapted cells) increases expression of 196 genes and reduces expression of 528. In Null-MEFs, after 24 h of high NaCl, expression of 217 genes increase and 428 decrease, while in adapted Null-MEFs 143 increase and 622 decrease. Fewer than 10% of genes are regulated in common between WT-and null-MEFs, indicating a profound difference in regulation of highNaCl induced genes induced by NFAT5 compared with those induced in the absence of NFAT5. Based on our findings we suggest a mechanism for this phenomenon, which had previously been unexplained. The NFAT5 DNA-binding motif (osmotic response element) is overrepresented in the vicinity of genes that NFAT5 upregulates, but not genes that it downregulates. We used Gene Ontology and manual curation to determine the function of the genes targeted by NFAT5, revealing many novel consequences of NFAT5 transcriptional activity.NFAT5; hypertonicity; GO analysis; osmotic; RNA HYPERTONICITY, WHICH IS INDUCED by high extracellular concentrations of solutes such as NaCl, can damage and even kill cells, but cells generally protect themselves by responses including accumulation of compatible organic osmolytes (reviewed in Ref.2). The highest interstitial concentrations of NaCl in mammals occur in their renal medullas, consequent to the operation of the urinary concentrating mechanism, but interstitial NaCl can be elevated in other tissues as well, although not nearly to as great an extent in the renal medulla.Hypertonicity decreases gene expression in general (30) but also increases expression of many genes (8). Nuclear factor of activated T cells-5 (NFAT5) (16, 22) is a transcription factor that increases expression of genes clearly involved in osmoprotection, but also of other genes in which the connection is not obvious (9). Previous screens identified many hypertonicity-induced genes, including those regulated by NFAT5. Most of those screens employed DNA microarrays, for example (17,20,24,31). Recently, the development of RNA sequencing (RNA-Seq) has provided a means for a more comprehensive profiling of gene expression than previous methods, including DNA microarrays (3).In the present study we employed RNA-Seq to provide a comprehensive, unbiased account of genes regulated by high NaCl in mouse embryonic fibroblast cells (MEFs). To distinguish which of the genes are regulated b...

The kidney‐specific expression of genes can be modulated by the extracellular osmolality

“…Besides Prss35, the expression of these factors was not detected in renal segments (1). However, these transcripts have been identified to be up‐regulated by hyperosmolality in MEFs (37). Publicly available databases indicated that these genes physiologically have a kidney‐specific expression pattern (Table 2).…”

Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5

“…For example, by RNA-Seq we recently identified hundreds of NFAT5 transcriptional targets in mouse embryo fibroblast cells (30). Transcriptional regulation by NFAT5 is the obvious explanation for the effect it might have on activity of any protein.…”

Deletion of Von Hippel–Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern

“…Other prominent genes are the ran-binding protein 3 like (RanBP3L) or the ring finger 183 (Rnf183). The expression of both genes is induced by hyperosmolality [15,38,39]. However, in Vhl-KO cells, the expression of both genes is significantly downregulated.…”

“…When we compare the changes in expression, we observed that Vhl-KO cells showed reduced expression of 33 unfavorable and induced expression of 22 favorable genes. But the upregulated expression of more unfavorable genes (56) and predominantly reduced expression of favorable genes (38) indicates that, in summary, the loss of functional Vhl in the mpkCCD cells induces an unfavorable gene expression pattern.…”