Modeling bidirectional transcription using silkmoth chorion gene promoters.

Bidirectional transcription is an interesting feature of eukaryotic genomes; yet not all aspects of its mechanism are understood. Silkmoth choriogenesis is a model system for studying transcriptional regulation at the initiation level. As chorion genes comprise a large group of divergently transcribed gene pairs, we are presented with the possibility of investigating the intricacies of bidirectional transcription. Their well characterized 5' regulatory regions and expression profiles lay the foundation for investigating protein:protein and protein:DNA interactions, and RNA polymerase function during oocyte development. In this article we summarize current knowledge on chorion gene regulation and propose an approach to modeling bidirectional transcription using chorion promoters.

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 BmGATAbeta binding modes on a chorion gene promoter from the Er1.A/B early gene pair.

CHD1 assumes a central role during follicle development.

During Bombyx mori follicle development, fine-tuning of chorion gene expression is under the control of bidirectional promoters. In this work, we show that the silkmoth chromo-helicase/ATPase-DNA binding protein 1 (CHD1) ortholog is responsible for repositioning of nucleosomes on chorion promoters, where the factor binds specifically. Chorion genes, occupying a single chromosomal locus, rely on an almost identical set of cis elements for their differential expression. As a direct consequence of remodeling, interaction of C/EBP and TFIID with promoter elements is facilitated and ultimately leads to initiation of transcription. Appending of methylation marks to H3K4 in a temporal-specific manner is dependent on CHD1 binding to cognate cis elements and signifies gene activation. Overall, CHD1 is a critical factor for proper development of the follicular epithelium in terms of whole-cell chromatin arrangement.

Architectural factor HMGA induces promoter bending and recruits C/EBP and GATA during silkmoth chorion gene regulation.

A protein displaying significant similarity to mammalian HMGA (high-mobility group A) proteins, but also bearing unique structural features, was isolated from silkmoth (Bombyx mori) follicular cells. This factor, named BmHMGA, exhibits specific binding preference for chorion gene promoter elements and induces DNA bending thereon. BmHMGA deploys temporal-specific interaction with transcription factors BmC/EBP (C/EBP is CCAAT/enhancer-binding protein) and BmGATAbeta during follicle maturation. The respective protein complexes can be detected on chorion gene promoters in vivo, with different developmental profiles each time. Analogous interaction takes place on the putative promoter of the BmC/EBP gene, hinting towards a transcriptional circuit that is responsible for the progress of choriogenesis as a whole. Finally, transient suppression of BmHMGA expression led to down-regulation of chorion genes and the BmC/EBP gene, and revealed recruitment of BmC/EBP, BmGATAbeta and TFIID (transcription factor IID)/TBP (TATA-box-binding protein) by BmHMGA. A revised model for chorion gene regulation is discussed in view of these findings.

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

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.

Inference of evolutionary patterns of the land snail Albinaria in the Aegean archipelago: is vicariance enough?

Mitochondrial DNA sequences from 16S rRNA and ATPase8 genes were used to investigate phylogeographic patterns of the land snail Albinaria (Gastropoda: Clausiliidae) in the Aegean archipelago. Forty-two populations of Albinaria were analyzed, mainly A. turrita, A. caerulea and A. brevicollis, collected from 22 Aegean islands and certain surrounding regions. Maximum parsimony, maximum likelihood and Bayesian analyses on 16S rRNA and combined datasets produced trees that share significant similarity and reveal a phylogeny with distinct branches which are in general, but not full, agreement with current taxonomy. The Aegean taxa are not monophyletic as a whole, since A. turrita does not cluster with A. caerulea and A. brevicollis. The latter form a distinct monophyletic cluster, within which two groups are evident. These groups do not readily correspond to currently accepted morphospecies; one contains the populations that inhabit the central part of the archipelago plus some eastern islands, while the other contains populations whose geographic distribution is restricted to the southeastern part of the archipelago. The divergence between these two groups is attributed to vicariance events that primarily shape contemporary distributions. Although dispersal may also be present, certain small- and large-scale vicariance events can be traced; alternative phylogeographic hypotheses are discussed in view of the historical biogeography of the region.

MITOCHONDRIAL PHYLOGEOGRAPHY OF THE LAND SNAIL ALBINARIA IN CRETE: LONG-TERM GEOLOGICAL AND SHORT-TERM VICARIANCE EFFECTS.

The land snail genus Albinaria exhibits an extreme degree of morphological differentiation in Greece, especially in the island of Crete. Twenty-six representatives of 17 nominal species and a suspected hybrid were examined by sequence analysis of a PCR-amplified mitochondrial DNA fragment of the large rRNA subunit gene. Maximum parsimony and neighbor-joining phylogenetic analyses demonstrate a complex pattern of speciation and differentiation and suggest that Albinaria species from Crete belong to at least three distinct monophyletic groups, which, however, are not monophyletic with reference to the genus as a whole. There is considerable variation of genetic distance within and among "species" and groups. The revealed phylogenetic relations do not correlate well with current taxonomy, but exhibit biogeographical coherence. Certain small- and large-scale vicariance events can be traced, although dispersal and parapatric speciation may also be present. Our analysis suggests that there was an early and rapid differentiation of Albinaria groups across the whole of the range followed by local speciation events within confined geographical areas.