AOKI MARIA DEL PILAR
Congresos y reuniones científicas
Título:
Expression of the ZFHEP gene during neurogenensis and myogenesis
Autor/es:
ROQUEIRO G; MANAVELLA P; AOKI MP; GEA S; DARLING D; CABANILLAS AM
Reunión:
Congreso; XL Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecula; 2004
Resumen:
Zfhep (Zinc Finger Homeodomain Enhancer-binding Protein) is a
transcription factor involved in differentiation processes such as
myogenesis, neurogenesis and lymphopoiesis. The promoter has
been recently isolated and so far, there is no direct assessment of
its regulation during cell differentiation. Our goal was to evaluate
the activity of Zfhep promoter during myocyte and neuron
development. The embryonal carcinoma (P19) and a mouse
myoblast (C2C12) cell lines were differentiated for inducing
neurogenesis (by pretreatment with retinoic acid) or myogenesis
(by incubation with 2% horse serum), respectively. Western blot
and immunocytochemistry methods were used to validate the
model. Human Zfhep promoter was cloned into pGL3-basic and
several mutant deletions were made. The reporter chimeras and
CMVβ-gal (to normalize transfection efficiency) were transfected
into C2C12 and P19 cells by calcium phosphate and FuGENE6,
respectively; then they were induced to differentiate. Luciferase
and β-galactosidase activities were assessed in cell lysates by
standard methods. The promoter was active in all of the constructs
tested in both, undifferentiated C2C12 and P19 cells. The activity
of the Zfhep promoter, decreased significantly after differentiation
of P19 to neurons. The activity was also lowered by 30 fold in
differentiated C2C12 cells. These results first show that the Zfhep
gene is dynamically regulated during myogenesis and neurogenesis.
The repression of the gene could be related to a lack of function of
Zfhep during the differentiated cell state.
transcription factor involved in differentiation processes such as
myogenesis, neurogenesis and lymphopoiesis. The promoter has
been recently isolated and so far, there is no direct assessment of
its regulation during cell differentiation. Our goal was to evaluate
the activity of Zfhep promoter during myocyte and neuron
development. The embryonal carcinoma (P19) and a mouse
myoblast (C2C12) cell lines were differentiated for inducing
neurogenesis (by pretreatment with retinoic acid) or myogenesis
(by incubation with 2% horse serum), respectively. Western blot
and immunocytochemistry methods were used to validate the
model. Human Zfhep promoter was cloned into pGL3-basic and
several mutant deletions were made. The reporter chimeras and
CMVβ-gal (to normalize transfection efficiency) were transfected
into C2C12 and P19 cells by calcium phosphate and FuGENE6,
respectively; then they were induced to differentiate. Luciferase
and β-galactosidase activities were assessed in cell lysates by
standard methods. The promoter was active in all of the constructs
tested in both, undifferentiated C2C12 and P19 cells. The activity
of the Zfhep promoter, decreased significantly after differentiation
of P19 to neurons. The activity was also lowered by 30 fold in
differentiated C2C12 cells. These results first show that the Zfhep
gene is dynamically regulated during myogenesis and neurogenesis.
The repression of the gene could be related to a lack of function of
Zfhep during the differentiated cell state.
