Joung-Woo Hong

Inhibition of NF-κB by ZAS3, a zinc-finger protein that also binds to the κB motif

The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the κB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The κB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-κB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the κB motif during acute-phase responses. A functional link between ZAS and NF-κB, two distinct families of κB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-κB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major κB-binding proteins in B lymphocytes, and that NF-κB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-κB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for κB gene regulatory elements, and repression of target gene transcription.

ZAS3 represses NFκB-dependent transcription by direct competition for DNA binding

NFκB and ZAS3 are transcription factors that control important cellular processes including immunity, cell survival and apoptosis. Although both proteins bind the κB-motif, they produce opposite physiological consequences; NFκB activates transcription, promotes cell growth and is often found to be constitutively expressed in cancer cells, while ZAS3 generally represses transcription, inhibits cell proliferation and is downregulated in some cancers. Here, we show that ZAS3 inhibits NFκB-dependent transcription by competing with NFκB for the κB-motif. Transient transfection studies show that N-terminal 645 amino acids is sufficient to repress transcription activated by NFκB, and that the identical region also possesses intrinsic repression activity to inhibit basal transcription from a promoter. Finally, in vitro DNA-protein interaction analysis shows that ZAS3 is able to displace NFκB by competing with NFκB for the κB-motif. It is conceivable that ZAS3 has therapeutic potential for controlling aberrant activation of NFκB in various diseases.

Midline enhancer activity of the short gastrulation shadow enhancer is characterized by three unusual features for cis-regulatory DNA

The shadow enhancer of the short gastrulation (sog) gene directs its sequential expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo. Here, we characterize three unusual features of the shadow enhancer midline activity. First, the minimal regions for the two different enhancer activities exhibit high overlap within the shadow enhancer, meaning that one developmental enhancer possesses dual enhancer activities. Second, the midline enhancer activity relies on five Single-minded (Sim)-binding sites, two of which have not been found in any Sim target enhancers. Finally, two linked Dorsal (Dl)- and Zelda (Zld)-binding sites, critical for the neurogenic ectoderm enhancer activity, are also required for the midline enhancer activity. These results suggest that early activation by Dl and Zld may facilitate late activation via the noncanonical sites occupied by Sim. We discuss a model for Zld as a pioneer factor and speculate its role in midline enhancer activity. [BMB Reports 2015; 48(10): 589-594]

Capicua is involved in Dorsal-mediated repression of zerknüllt expression in Drosophila embryo.

The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. Here, we present evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknüllt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressorbinding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE. [BMB Reports 2014; 47(9):518-523]

Transcriptional activity of the short gastrulation primary enhancer in the ventral midline requires its early activity in the presumptive neurogenic ectoderm.

The short gastrulation (sog) shadow enhancer directs early and late sog expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo, respectively. Here, evidence is presented that the sog primary enhancer also has both activities, with the late enhancer activity dependent on the early activity. Computational analyses showed that the sog primary enhancer contains five Dorsal (Dl)-, four Zelda (Zld)-, three Bicoid (Bcd)-, and no Single-minded (Sim)-binding sites. In contrast to many ventral midline enhancers, the primary enhancer can direct lacZ expression in the ventral midline as well as in the neurogenic ectoderm without a canonical Simbinding site. Intriguingly, the impaired transcriptional synergy between Dl and either Zld or Bcd led to aberrant and abolished lacZ expression in the neurogenic ectoderm and in the ventral midline, respectively. These findings suggest that the two enhancer activities of the sog primary enhancer are functionally consolidated and geographically inseparable. [BMB Reports 2016; 49(10): 572-577]