Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements

https://doi.org/10.1016/j.bbrc.2008.02.113Get rights and content

Abstract

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

Section snippets

Materials and methods

Southwestern assays. Staged nuclear extracts, derived from dissection of 100 pupae of the FengIx54A strain, were gently heated at 37 °C and electrophoresed in a 12% SDS–PAGE gel (50 μg in standard 1× sample buffer lacking SDS) at 4 °C. Proteins were subsequently transferred on nitrocellulose (Schleicher&Schuell) using the TE70 Semiphor unit (Pharmacia). After the transfer the membrane was washed once in TNT buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and once in 1× Binding Buffer

Results and discussion

C/EBP recognition sequences have been previously identified in chorion gene promoters; these were classified as early- or late-type, depending on a subtle variation in sequence [8]. Their frequency of occurrence on promoters of different developmental specificity follows a more or less simple pattern: early-type sites predominate in early and early-middle genes (i.e. ErA/B.1, 6F6.2, A/B.L9) and their gradual replacement by late-type sites can be observed in middle-late (i.e. A/B.L1) and late

Acknowledgments

Authors are indebted to P. Rørth for providing the C135 antiserum. This work was supported by a GSRT grant (03ED124) funded by the E.U. and the Greek government. A.P. is the recipient of an SSF personal scholarship for postgraduate studies.

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  • BmVMP90, a large vitelline membrane protein of the domesticated silkmoth Bombyx mori, is an essential component of the developing ovarian follicle

    2012, Insect Biochemistry and Molecular Biology
    Citation Excerpt :

    Significant progress has been reported with respect to the regulation of the expression of chorion genes in Bombyx (Lecanidou and Papantonis, 2010). Transcription factors that regulate the transition from vitellogenesis to choriogenesis include the orphan nuclear receptor BmE75C (Georgomanolis et al., 2009; Swevers et al., 2002), the BmGATAβ zinc finger protein and the BmC/EBP factor (Drevet et al., 1994, 1995; Papantonis et al., 2008; Sourmeli et al., 2003). To clarify the role of BmGATAβ during choriogenesis, hybridization screens aimed at the identification of differentially expressed genes during oogenesis were carried out (Kendirgi et al., 2002) as well as yeast two-hybrid screens for the isolation of interacting proteins (Glushek, 2001).

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Present address: Pharmaserve-Lilly S.A.C.I., 14564 Kifissia, Greece.

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