A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis

One previously unidentified cDNA clone coding for a C/EBP factor, BmC/EBP, was isolated from Bombyx mori follicular cells. This is the first time that a C/EBP factor has been isolated and characterized in Lepidoptera. We provide information concerning structural features and developmental specificity, as well as in vitro interaction properties with chorion gene promoter modules. BmC/EBP was capable of effectively recognizing homologous binding sites from chorion gene promoters derived from flies and other moths, despite significant diversity of chorion structure, gene organization, and gene expression profiles. We propose that the relative concentration of BmC/EBP, in relation to its differential binding affinity for promoter cis-elements, results in activation or repression of silkmoth chorion gene expression.     

BmCbZ, an insect-specific factor featuring a composite DNA-binding domain, interacts with BmC/EBPgamma

A novel factor featuring a composite AT hook/basic region-leucine zipper DNA-binding domain was isolated from Bombyx mori follicular cells. Screening of EST databases derived from a variety of metazoans revealed the exclusive presence of BmCbZ homologues in insect species. BmCbZ characteristic features and gene organization are discussed, in comparison to other known bZIP factors. We concordantly propose that this factor establishes a new insect-specific bZIP family. We further present the isolation of the silkmoth homologue of mammalian C/EBPgamma, BmC/EBPgamma, and in vitro evidence for its interaction with BmCbZ. The formation of a BmCbZ-BmC/EBPgamma heterodimer is a prerequisite for binding to specific C/EBP recognition sites on chorion gene promoters, most probably via both major and minor groove interactions.     

A modified chromatin-immunoprecipitation protocol for silkmoth ovarian follicular cells reveals C/EBP and GATA binding modes on an early chorion gene promoter

Standard Chromatin immunoprecipitation protocols have been designed to suit studies performed on cell line cultures or yeast cells growing in liquid cultures. In these cases cross-linking/fixation takes place directly in the growing medium of the cells by the addition of a general fixation reagent. When applied on whole isolated silkmoth follicles, this procedure results in poor release of follicular cells from the basal membrane and lower yield of cross-linked chromatin. We present a modification to the standard protocol, where detachment of follicular cells from the basal membrane of the egg and nuclei isolation precedes formaldehyde-mediated cross-linking. We also discuss application of the modified method for the identification of distinct BmC/EBP and BmGATAβ binding modes on a chorion gene promoter from the Er1.A/B early gene pair.     

Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.     

In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors

This is the first attempt to identify regulatory elements that are involved in early choriogenesis of the silkworm Bombyx mori. A new cis element in the promoter region of five early chorion genes was identified. The consensus sequence of this element matches the consensus of the C/EBP DNA binding site. Moreover, this sequence interacts with a 70 kD protein (pX2) present in follicular nuclear extracts and complex formation exhibits early developmental specificity. There is strong evidence that this factor belongs to the C/EBP family. Surprisingly, the same protein binds with the same developmental specificity to a similar sequence of a late chorion gene promoter, which has been previously defined as the binding site for a putative late specific factor, BCFII. The possibility that pX2 and BCFII are isoforms or modifications of the same protein factor, which is presumably able to bind to the highly similar sequence elements of both early and late genes, is discussed. A hypothesis involving protein-protein interactions between C/EBP (pX2/BCFII) and GATA during choriogenesis is presented to explain the temporal specificity of chorion genes.     

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive.     

The silkmoth eggshell as a natural amyloid shield for the safe development of insect oocyte and embryo: insights from studies of silkmoth chorion protein peptide-analogues of the B family

Silkmoth chorion is the major component of the silkmoth eggshell. The proteins that constitute more than 95% of its dry mass have remarkable mechanical and physicochemical properties forming a protective natural shield for the oocyte and the developing embryo from a wide range of environmental hazards. Peptide-analogues of the central conservative domain of the two major families of silkmoth chorion proteins, the A's and the B's, form amyloid fibrils under a variety of conditions, which prompted us to propose, 10 years ago, that silkmoth chorion is an amyloid with protective properties. Following our finding, a number of studies verified the existence of several functional amyloids. In this study, we designed, synthesized and studied two peptide-analogues of the central conservative domain of the B family of silkmoth chorion proteins, and we present experimental results, which show: (a) that the amyloidogenic properties of silkmoth chorion peptides are encoded into the tandemly repeating hexapeptides comprising the central domain of silkmoth chorion proteins, confirming our previous findings from peptide analogues of the A family of chorion proteins, and, (b) they suggest how silkmoth chorion proteins of the B family self-assemble in vivo, for the formation of the helicoidal architecture of silkmoth chorion.     

Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145].