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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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Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.
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pone-0004471-g005: Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.

Mentions: To investigate if Foxi1 regulates expression of the a4 subunit we studied the a4 promoter sequence and identified a cluster of three potential forkhead binding cis-elements (Fk1-3) adhering to the forkhead binding consensus sequence (Fig. 4; T(g/a)TTT(g/a) (t/c); [14]). Reporter constructs encompassing these sites show a dose-dependent Foxi1 activation (Fig. 4). Some two hundred bases downstream from this cluster we found another potential forkhead binding site (Fk4; Fig. 4). When any of the three sites in the cluster were mutated there was a significant drop in reporter gene activity, the most prominent effect was noted for the Fk3 site (Fig. 5A, B). While mutations at this site essentially blocked Foxi1 dependent inducibility the Fk2 site reduced Foxi1 ability to trans-activate by approximately half and finally Fk3 reduced this effect by about a third (Fig. 5A, B). Mutations at Fk4 did not significantly affect reporter activity. In EMSA experiments we could demonstrate a distinct mobility shift using a labeled double stranded oligonucleotide harboring the Fk1-3 cluster (Fig. 5C). We could also observe a much weaker interaction for Fk4 (Fig. 5C) that agrees well with the effects these sites have on total Foxi1 mediated reporter gene activation (Fig. 5B). To investigate the nature of these interactions we performed a series of EMSA experiments using the same mutations as in the transfection experiments and wt (wild type) sequences (Fig. 5D–F). While it can be deduced from figures 5D, E and F that the unlabeled wt probe effectively competes for the Fk1, 2 and 3 protein interactions at concentrations of approximately 10-fold molar excess, compatible with bona fide interactions, the mutated Fk1, 2 and 3 probes are significantly less efficient as competitors and display significant competition only at ∼100-fold molar excess in most instances, a finding that agrees well with transfection data (Fig. 5B). Taken together, these results suggest that Foxi1 is able to interact with the ATP6V0A4 promoter at position −561/−547 in a sequence specific manner and this argues in favor for direct Foxi1-mediated regulation of ATP6V0A4 gene expression in kidney, inner ear and epididymis. We have previously shown in transfection experiments that FOXI1 interacts with a key cis-element in the ATP6V1B1 promoter, at −102/−96, and that this interaction is required for a dose-dependent activation by FOXI1 [13]. In conclusion, this is compatible with a direct role for Foxi1 in transcriptional regulation of a4 and B1 subunits and establishes these genes as Foxi1 downstream targets. In chromatin immunoprecipitation experiments, aimed to study in vivo interactions at the Fk1-3 cluster, we confirm that Foxi1 interactions are strongest at Fk3, slightly weaker at Fk2 and weakest at Fk1 (Fig. 6). When all three sites in the Fk1-3 cluster are mutated we cannot detect any Foxi1 interaction (Fig. 6). An in silico analysis of upstream regions in the A1 and E2 genes demonstrates the presence of several potential forkhead binding sites conforming to the consensus sequence t(g/a)TTT(g/a)(t/c) (Overdier et al., 1997; Fig. 7).


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)

Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.
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pone-0004471-g005: Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.
Mentions: To investigate if Foxi1 regulates expression of the a4 subunit we studied the a4 promoter sequence and identified a cluster of three potential forkhead binding cis-elements (Fk1-3) adhering to the forkhead binding consensus sequence (Fig. 4; T(g/a)TTT(g/a) (t/c); [14]). Reporter constructs encompassing these sites show a dose-dependent Foxi1 activation (Fig. 4). Some two hundred bases downstream from this cluster we found another potential forkhead binding site (Fk4; Fig. 4). When any of the three sites in the cluster were mutated there was a significant drop in reporter gene activity, the most prominent effect was noted for the Fk3 site (Fig. 5A, B). While mutations at this site essentially blocked Foxi1 dependent inducibility the Fk2 site reduced Foxi1 ability to trans-activate by approximately half and finally Fk3 reduced this effect by about a third (Fig. 5A, B). Mutations at Fk4 did not significantly affect reporter activity. In EMSA experiments we could demonstrate a distinct mobility shift using a labeled double stranded oligonucleotide harboring the Fk1-3 cluster (Fig. 5C). We could also observe a much weaker interaction for Fk4 (Fig. 5C) that agrees well with the effects these sites have on total Foxi1 mediated reporter gene activation (Fig. 5B). To investigate the nature of these interactions we performed a series of EMSA experiments using the same mutations as in the transfection experiments and wt (wild type) sequences (Fig. 5D–F). While it can be deduced from figures 5D, E and F that the unlabeled wt probe effectively competes for the Fk1, 2 and 3 protein interactions at concentrations of approximately 10-fold molar excess, compatible with bona fide interactions, the mutated Fk1, 2 and 3 probes are significantly less efficient as competitors and display significant competition only at ∼100-fold molar excess in most instances, a finding that agrees well with transfection data (Fig. 5B). Taken together, these results suggest that Foxi1 is able to interact with the ATP6V0A4 promoter at position −561/−547 in a sequence specific manner and this argues in favor for direct Foxi1-mediated regulation of ATP6V0A4 gene expression in kidney, inner ear and epididymis. We have previously shown in transfection experiments that FOXI1 interacts with a key cis-element in the ATP6V1B1 promoter, at −102/−96, and that this interaction is required for a dose-dependent activation by FOXI1 [13]. In conclusion, this is compatible with a direct role for Foxi1 in transcriptional regulation of a4 and B1 subunits and establishes these genes as Foxi1 downstream targets. In chromatin immunoprecipitation experiments, aimed to study in vivo interactions at the Fk1-3 cluster, we confirm that Foxi1 interactions are strongest at Fk3, slightly weaker at Fk2 and weakest at Fk1 (Fig. 6). When all three sites in the Fk1-3 cluster are mutated we cannot detect any Foxi1 interaction (Fig. 6). An in silico analysis of upstream regions in the A1 and E2 genes demonstrates the presence of several potential forkhead binding sites conforming to the consensus sequence t(g/a)TTT(g/a)(t/c) (Overdier et al., 1997; Fig. 7).

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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