Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

Abstract

BackgroundCabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein.Methodology/Principal FindingsTo determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2.Conclusions/SignificanceOur results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins.

Cabut/ dTIEG associates with the transcription factor Yorkie for growth control

“…Gene Ontology (GO) analysis indicated that this subset of genes was enriched in transcriptional activity, cell migration, mitotic cell cycle and signaling pathways known to play a role in imaginal disk development (Supplementary Fig S1B). As expected, among Cbt targets we found previously described genes such as salm ( spalt major ) or cbt itself , but also several unidentified target genes such as wg ( wingless ) or vg ( vestigial ) (Fig B).…”

“…Control samples (mock) of GFP À Marina Ruiz-Romero et al Cabut/dTIEG is a Yorkie partner for growth control EMBO reports subset of genes was enriched in transcriptional activity, cell migration, mitotic cell cycle and signaling pathways known to play a role in imaginal disk development ( Supplementary Fig S1B). As expected, among Cbt targets we found previously described genes such as salm (spalt major) or cbt itself [11,17], but also several unidentified target genes such as wg (wingless) or vg (vestigial) ( Fig 1B). Cbt association around the TSS may be an indication of its function as a primary regulatory element, but does not provide any information about its role as an activator or a repressor.…”

“…A recent study identified a novel Mad‐like motif in promoters of Cbt‐regulated genes . However, this new motif does not coincide with previously reported Cbt binding data .…”

“…Taken together, our results suggest that Cbt functions as a transcriptional activator in the wing disk. Nevertheless, we cannot discard its contribution to repression in some contexts or through binding to different partners, as previous experiments have demonstrated the relevance of the Sin3A interaction domain for Cbt's repressive role [17].…”

New Role for Kruppel-like Factor 14 as a Transcriptional Activator Involved in the Generation of Signaling Lipids

“…Moreover, select functions of some of these proteins (such as KLF11 and KLF14) can cause common human metabolic diseases, including neonatal and juvenile diabetes, as well as the obesity-insulin resistance-metabolic syndrome, which leads to adult type 2 diabetes (10,16,17). Thus, because of their structural conservation and function, these six proteins have become to be known as the Cabut family of metabolic regulators (9,18). Consequently, we and others have recently focused on how these proteins regulate the expression of genes and gene networks involved in lipid metabolism and signaling.…”