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Essential role of the N-terminal region of TFII-I in viability and behavior.

Lucena J, Pezzi S, Aso E, Valero MC, Carreiro C, Dubus P, Sampaio A, Segura M, Barthelemy I, Zindel MY, Sousa N, Barbero JL, Maldonado R, Pérez-Jurado LA, Campuzano V - BMC Med. Genet. (2010)

Bottom Line: Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs.Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players.In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics Unit, de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT

Background: GTF2I codes for a general intrinsic transcription factor and calcium channel regulator TFII-I, with high and ubiquitous expression, and a strong candidate for involvement in the morphological and neuro-developmental anomalies of the Williams-Beuren syndrome (WBS). WBS is a genetic disorder due to a recurring deletion of about 1,55-1,83 Mb containing 25-28 genes in chromosome band 7q11.23 including GTF2I. Completed homozygous loss of either the Gtf2i or Gtf2ird1 function in mice provided additional evidence for the involvement of both genes in the craniofacial and cognitive phenotype. Unfortunately nothing is now about the behavioral characterization of heterozygous mice.

Methods: By gene targeting we have generated a mutant mice with a deletion of the first 140 amino-acids of TFII-I. mRNA and protein expression analysis were used to document the effect of the study deletion. We performed behavioral characterization of heterozygous mutant mice to document in vivo implications of TFII-I in the cognitive profile of WBS patients.

Results: Homozygous and heterozygous mutant mice exhibit craniofacial alterations, most clearly represented in homozygous condition. Behavioral test demonstrate that heterozygous mutant mice exhibit some neurobehavioral alterations and hyperacusis or odynacusis that could be associated with specific features of WBS phenotype. Homozygous mutant mice present highly compromised embryonic viability and fertility. Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs.

Conclusion: Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players. In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

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Growth properties of Gtf2iΔex2 mutant MEFs. A. Spontaneous immortalization. Eleven independent MEF cultures showing early immortalization in Gtf2iΔex2/Δex2 and later in Gtf2i+/Δex2 cells. B. Saturation rate. 106 cells plated by triplicate and counted every three days until they reached the maximum score. A poorer saturation rate was observed in Gtf2i+/Δex2 cells. C. Proliferation of immortal MEFs. Three independent experiments were performed with all cell types and each time point was done by triplicate. The fastest proliferation was observed in Gtf2iΔex2/Δex2 while Gtf2i+/Δex2 cells performed the worst. D. Re-entry into S-phase after serum deprivation. The percentage of cells in S-phase was measured at the indicated times after serum stimulation. Again Gtf2i+/Δex2 MEFs showed the lowest rate of S-phase re-entry. Gtf2i+/+, open rhombus; Gtf2i+/Δex2 grey squares; Gtf2iΔex2/Δex2 black triangles. All values are expressed as mean ± s.d.
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Figure 2: Growth properties of Gtf2iΔex2 mutant MEFs. A. Spontaneous immortalization. Eleven independent MEF cultures showing early immortalization in Gtf2iΔex2/Δex2 and later in Gtf2i+/Δex2 cells. B. Saturation rate. 106 cells plated by triplicate and counted every three days until they reached the maximum score. A poorer saturation rate was observed in Gtf2i+/Δex2 cells. C. Proliferation of immortal MEFs. Three independent experiments were performed with all cell types and each time point was done by triplicate. The fastest proliferation was observed in Gtf2iΔex2/Δex2 while Gtf2i+/Δex2 cells performed the worst. D. Re-entry into S-phase after serum deprivation. The percentage of cells in S-phase was measured at the indicated times after serum stimulation. Again Gtf2i+/Δex2 MEFs showed the lowest rate of S-phase re-entry. Gtf2i+/+, open rhombus; Gtf2i+/Δex2 grey squares; Gtf2iΔex2/Δex2 black triangles. All values are expressed as mean ± s.d.

Mentions: Next, we decided to test whether Δ140TFII-I may modify the direct link between mitogen-dependent signaling and changes in nuclear gene expression that govern cellular proliferation and cell division. Eleven independent fibroblast cultures derived from six different embryos were grown following the classical 3T3 protocol to obtain fully immortalized clones [16]. As illustrated in Figure 2A, Gtf2iΔex2/Δex2 MEF cultures immortalized around eight passages earlier than Gtf2i+/+ and Gtf2i+/Δex2 cultures independently of the chosen immortalization pathway (p53 dependent or not).


Essential role of the N-terminal region of TFII-I in viability and behavior.

Lucena J, Pezzi S, Aso E, Valero MC, Carreiro C, Dubus P, Sampaio A, Segura M, Barthelemy I, Zindel MY, Sousa N, Barbero JL, Maldonado R, Pérez-Jurado LA, Campuzano V - BMC Med. Genet. (2010)

Growth properties of Gtf2iΔex2 mutant MEFs. A. Spontaneous immortalization. Eleven independent MEF cultures showing early immortalization in Gtf2iΔex2/Δex2 and later in Gtf2i+/Δex2 cells. B. Saturation rate. 106 cells plated by triplicate and counted every three days until they reached the maximum score. A poorer saturation rate was observed in Gtf2i+/Δex2 cells. C. Proliferation of immortal MEFs. Three independent experiments were performed with all cell types and each time point was done by triplicate. The fastest proliferation was observed in Gtf2iΔex2/Δex2 while Gtf2i+/Δex2 cells performed the worst. D. Re-entry into S-phase after serum deprivation. The percentage of cells in S-phase was measured at the indicated times after serum stimulation. Again Gtf2i+/Δex2 MEFs showed the lowest rate of S-phase re-entry. Gtf2i+/+, open rhombus; Gtf2i+/Δex2 grey squares; Gtf2iΔex2/Δex2 black triangles. All values are expressed as mean ± s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865459&req=5

Figure 2: Growth properties of Gtf2iΔex2 mutant MEFs. A. Spontaneous immortalization. Eleven independent MEF cultures showing early immortalization in Gtf2iΔex2/Δex2 and later in Gtf2i+/Δex2 cells. B. Saturation rate. 106 cells plated by triplicate and counted every three days until they reached the maximum score. A poorer saturation rate was observed in Gtf2i+/Δex2 cells. C. Proliferation of immortal MEFs. Three independent experiments were performed with all cell types and each time point was done by triplicate. The fastest proliferation was observed in Gtf2iΔex2/Δex2 while Gtf2i+/Δex2 cells performed the worst. D. Re-entry into S-phase after serum deprivation. The percentage of cells in S-phase was measured at the indicated times after serum stimulation. Again Gtf2i+/Δex2 MEFs showed the lowest rate of S-phase re-entry. Gtf2i+/+, open rhombus; Gtf2i+/Δex2 grey squares; Gtf2iΔex2/Δex2 black triangles. All values are expressed as mean ± s.d.
Mentions: Next, we decided to test whether Δ140TFII-I may modify the direct link between mitogen-dependent signaling and changes in nuclear gene expression that govern cellular proliferation and cell division. Eleven independent fibroblast cultures derived from six different embryos were grown following the classical 3T3 protocol to obtain fully immortalized clones [16]. As illustrated in Figure 2A, Gtf2iΔex2/Δex2 MEF cultures immortalized around eight passages earlier than Gtf2i+/+ and Gtf2i+/Δex2 cultures independently of the chosen immortalization pathway (p53 dependent or not).

Bottom Line: Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs.Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players.In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics Unit, de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT

Background: GTF2I codes for a general intrinsic transcription factor and calcium channel regulator TFII-I, with high and ubiquitous expression, and a strong candidate for involvement in the morphological and neuro-developmental anomalies of the Williams-Beuren syndrome (WBS). WBS is a genetic disorder due to a recurring deletion of about 1,55-1,83 Mb containing 25-28 genes in chromosome band 7q11.23 including GTF2I. Completed homozygous loss of either the Gtf2i or Gtf2ird1 function in mice provided additional evidence for the involvement of both genes in the craniofacial and cognitive phenotype. Unfortunately nothing is now about the behavioral characterization of heterozygous mice.

Methods: By gene targeting we have generated a mutant mice with a deletion of the first 140 amino-acids of TFII-I. mRNA and protein expression analysis were used to document the effect of the study deletion. We performed behavioral characterization of heterozygous mutant mice to document in vivo implications of TFII-I in the cognitive profile of WBS patients.

Results: Homozygous and heterozygous mutant mice exhibit craniofacial alterations, most clearly represented in homozygous condition. Behavioral test demonstrate that heterozygous mutant mice exhibit some neurobehavioral alterations and hyperacusis or odynacusis that could be associated with specific features of WBS phenotype. Homozygous mutant mice present highly compromised embryonic viability and fertility. Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs.

Conclusion: Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players. In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

Show MeSH
Related in: MedlinePlus