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A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation.

Miyamoto Y, Taniguchi H, Hamel F, Silversides DW, Viger RS - BMC Mol. Biol. (2008)

Bottom Line: Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription.Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters.Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontogeny-Reproduction Research Unit, Centre de Recherche du Centre Hospitalier Universitaire de Quebec (CRCHUQ), Quebec City, QC, G1V 4G2, Canada. yokomiyamo@hotmail.com

ABSTRACT

Background: In mammals, sex determination is genetically controlled. The SRY gene, located on Y chromosome, functions as the dominant genetic switch for testis development. The SRY gene is specifically expressed in a subpopulation of somatic cells (pre-Sertoli cells) of the developing urogenital ridge for a brief period during gonadal differentiation. Despite this tight spatiotemporal expression pattern, the molecular mechanisms that regulate SRY transcription remain poorly understood. Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription.

Results: Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters. In the mouse, Wilms' tumor 1 (WT1) is an important regulator of both Sry and Müllerian inhibiting substance (Amh/Mis) expression and in humans, WT1 mutations are associated with abnormalities of sex differentiation. GATA4 transcriptionally cooperated with WT1 on the mouse, pig, and human SRY promoters. Maximal GATA4/WT1 synergism was dependent on WT1 but not GATA4 binding to their consensus regulatory elements in the SRY promoter and required both the zinc finger and C-terminal regions of the GATA4 protein. Although both isoforms of WT1 synergized with GATA4, synergism was stronger with the +KTS rather than the -KTS isoform. WT1/GATA4 synergism was also observed on the AMH promoter. In contrast to SRY, WT1/GATA4 action on the mouse Amh promoter was specific for the -KTS isoform and required both WT1 and GATA4 binding.

Conclusion: Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

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The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1. A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
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Figure 4: The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1. A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).

Mentions: To assess the binding site requirements for transcriptional synergism between GATA4 and WT1, mouse Sry promoter deletion and mutation constructs were prepared and used in co-transfection assays (Fig. 4A). Interestingly, the -340 bp Sry promoter construct (containing an intact proximal WT1 site and no consensus GATA sites) was synergistically activated by GATA4 and WT1 to the same extent as the full-length (-1090 bp) Sry construct. This suggests that GATA4 binding to its consensus sites on the Sry promoter is dispensable for maximal synergism with WT1. Deletion or mutation of the proximal WT1 site, however, abolished the activation by WT1 alone and markedly decreased the synergism between GATA4 and WT1 (Fig. 4A). Thus, in contrast to GATA4, binding of WT1 to its consensus element is critical for GATA4/WT1 synergism on the mouse Sry promoter. Interestingly, GATA4/WT1 synergism was not completely abrogated with the WT1 mutant constructs. The remaining synergism is likely due to two low affinity (not perfect consensus) GATA binding motifs present within the -340 to -70 bp sequence since a further deletion to -40 bp eliminated all WT1/GATA4 synergism (Fig. 4A). These two GATA motifs, named sites A (GTATCT) and B (GTATCT), are unique to the mouse Sry promoter sequence. Although these sites only weakly bind GATA4 protein (Fig. 4B), they are nonetheless functional as revealed by transfection assay (Fig. 4C). The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) was abolished when a truncated wild-type GATA4 protein (aa 201–440) was substituted with two GATA4 mutants (C294A or ΔT279) that we have previously shown to be unable to bind to DNA [54]. Thus, GATA4 binding to these two low affinity GATA motifs likely contributes to the observed GATA4/WT1 synergism on the mouse Sry promoter. We still cannot rule out the possibility, however, of additional unknown regulatory elements (present between -340 and -70 bp) that might be indirectly activated by GATA4/WT1 overexpression in our heterologous HeLa cell line model.


A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation.

Miyamoto Y, Taniguchi H, Hamel F, Silversides DW, Viger RS - BMC Mol. Biol. (2008)

The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1. A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1. A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a 32P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).
Mentions: To assess the binding site requirements for transcriptional synergism between GATA4 and WT1, mouse Sry promoter deletion and mutation constructs were prepared and used in co-transfection assays (Fig. 4A). Interestingly, the -340 bp Sry promoter construct (containing an intact proximal WT1 site and no consensus GATA sites) was synergistically activated by GATA4 and WT1 to the same extent as the full-length (-1090 bp) Sry construct. This suggests that GATA4 binding to its consensus sites on the Sry promoter is dispensable for maximal synergism with WT1. Deletion or mutation of the proximal WT1 site, however, abolished the activation by WT1 alone and markedly decreased the synergism between GATA4 and WT1 (Fig. 4A). Thus, in contrast to GATA4, binding of WT1 to its consensus element is critical for GATA4/WT1 synergism on the mouse Sry promoter. Interestingly, GATA4/WT1 synergism was not completely abrogated with the WT1 mutant constructs. The remaining synergism is likely due to two low affinity (not perfect consensus) GATA binding motifs present within the -340 to -70 bp sequence since a further deletion to -40 bp eliminated all WT1/GATA4 synergism (Fig. 4A). These two GATA motifs, named sites A (GTATCT) and B (GTATCT), are unique to the mouse Sry promoter sequence. Although these sites only weakly bind GATA4 protein (Fig. 4B), they are nonetheless functional as revealed by transfection assay (Fig. 4C). The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) was abolished when a truncated wild-type GATA4 protein (aa 201–440) was substituted with two GATA4 mutants (C294A or ΔT279) that we have previously shown to be unable to bind to DNA [54]. Thus, GATA4 binding to these two low affinity GATA motifs likely contributes to the observed GATA4/WT1 synergism on the mouse Sry promoter. We still cannot rule out the possibility, however, of additional unknown regulatory elements (present between -340 and -70 bp) that might be indirectly activated by GATA4/WT1 overexpression in our heterologous HeLa cell line model.

Bottom Line: Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription.Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters.Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontogeny-Reproduction Research Unit, Centre de Recherche du Centre Hospitalier Universitaire de Quebec (CRCHUQ), Quebec City, QC, G1V 4G2, Canada. yokomiyamo@hotmail.com

ABSTRACT

Background: In mammals, sex determination is genetically controlled. The SRY gene, located on Y chromosome, functions as the dominant genetic switch for testis development. The SRY gene is specifically expressed in a subpopulation of somatic cells (pre-Sertoli cells) of the developing urogenital ridge for a brief period during gonadal differentiation. Despite this tight spatiotemporal expression pattern, the molecular mechanisms that regulate SRY transcription remain poorly understood. Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription.

Results: Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters. In the mouse, Wilms' tumor 1 (WT1) is an important regulator of both Sry and Müllerian inhibiting substance (Amh/Mis) expression and in humans, WT1 mutations are associated with abnormalities of sex differentiation. GATA4 transcriptionally cooperated with WT1 on the mouse, pig, and human SRY promoters. Maximal GATA4/WT1 synergism was dependent on WT1 but not GATA4 binding to their consensus regulatory elements in the SRY promoter and required both the zinc finger and C-terminal regions of the GATA4 protein. Although both isoforms of WT1 synergized with GATA4, synergism was stronger with the +KTS rather than the -KTS isoform. WT1/GATA4 synergism was also observed on the AMH promoter. In contrast to SRY, WT1/GATA4 action on the mouse Amh promoter was specific for the -KTS isoform and required both WT1 and GATA4 binding.

Conclusion: Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

Show MeSH
Related in: MedlinePlus