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The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis.

Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, Enerbäck S - PLoS ONE (2009)

Bottom Line: Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner.Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter.Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

View Article: PubMed Central - PubMed

Affiliation: Center of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, Göteborg University, Göteborg, Sweden.

ABSTRACT
The vacuolar H(+)-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

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mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.Combined in situ hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.
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pone-0004471-g008: mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.Combined in situ hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.

Mentions: While overlapping expression patterns of the transcription factor Foxi1 and the a4 and B1 subunits to some degree is suggestive of a regulative role for Foxi1 it is somewhat surprising that the ubiquitously expressed subunits A1 and E2 also appear to be dependent on Foxi1 expression (Fig. 1–3). Although proper assembly has been shown to be important for both intracellular targeting/localization and stability of subunits [15], [16], [17] specific regulation of ubiquitously expressed genes/proteins constitutes another possible mode of regulation. To address the general question: whether this is due to an Foxi1 mediated effect on mRNA levels or if lack of the tissue specific subunits B1 and a4 would lead to destabilization of the multimeric complex and as a consequence hereof enhanced degradation of its constituents. We performed mRNA in situ hybridization on kidney sections from wt and Foxi1 −/− mice using mRNA anti-sense probes for A1 and E2. On the same sections, as described previously [12] we performed IHC using an antibody directed against carbonic anhydrase II (CAII) a marker for intercalated cells in the kidney (Fig. 8). While CAII positive intercalated cells are present in both wt and Foxi1−/− kidneys A1 and E2 mRNA can only be detected in wt kidneys. Thus, even though A1 and E2 belong to a class of more broadly expressed subunits they appear to require Foxi1 for expression of their mRNA in these cell types.


The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis.

Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, Enerbäck S - PLoS ONE (2009)

mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.Combined in situ hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637605&req=5

pone-0004471-g008: mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.Combined in situ hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.
Mentions: While overlapping expression patterns of the transcription factor Foxi1 and the a4 and B1 subunits to some degree is suggestive of a regulative role for Foxi1 it is somewhat surprising that the ubiquitously expressed subunits A1 and E2 also appear to be dependent on Foxi1 expression (Fig. 1–3). Although proper assembly has been shown to be important for both intracellular targeting/localization and stability of subunits [15], [16], [17] specific regulation of ubiquitously expressed genes/proteins constitutes another possible mode of regulation. To address the general question: whether this is due to an Foxi1 mediated effect on mRNA levels or if lack of the tissue specific subunits B1 and a4 would lead to destabilization of the multimeric complex and as a consequence hereof enhanced degradation of its constituents. We performed mRNA in situ hybridization on kidney sections from wt and Foxi1 −/− mice using mRNA anti-sense probes for A1 and E2. On the same sections, as described previously [12] we performed IHC using an antibody directed against carbonic anhydrase II (CAII) a marker for intercalated cells in the kidney (Fig. 8). While CAII positive intercalated cells are present in both wt and Foxi1−/− kidneys A1 and E2 mRNA can only be detected in wt kidneys. Thus, even though A1 and E2 belong to a class of more broadly expressed subunits they appear to require Foxi1 for expression of their mRNA in these cell types.

Bottom Line: Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner.Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter.Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

View Article: PubMed Central - PubMed

Affiliation: Center of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, Göteborg University, Göteborg, Sweden.

ABSTRACT
The vacuolar H(+)-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

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