cChromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ϳ700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β 1 -subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β 1 -mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β 1 , the surface expression of the β 1 -subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase β 1 -subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase β 1 -subunit attenuated TGF-β 1 -mediated EMT. We further show that both Na,K-ATPase α-and β-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-β 1 -mediated EMT and is associated with the progression of EMT in cancer and fibrosis. Mol Cancer Ther; 9(6); 1515-24. ©2010 AACR.

BackgroundInsulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man.ResultsIn the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF–CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity.ConclusionsOur results indicate that dCTCF–CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF–dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0168-7) contains supplementary material, which is available to authorized users.