Dervla M. Mellerick

Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator

At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861–873; Weiss et al. (1998) Genes Dev., 12, 3591–3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165–1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions.

Nk6, a novel Drosophila homeobox gene regulated by vnd

Nk(x)-type homeobox genes are an evolutionarily conserved family that regulate diverse developmental processes. Here we describe a novel Drosophila gene, Nk6, which encodes an Nk-type transcription factor most homologous to vertebrate Nkx6.1 and Nkx6.2. The homeodomains and NK decapeptide domains of all three proteins are highly conserved. Nk6 is expressed in the embryonic brain, ventral nerve cord, hindgut, and internal head structures. Nerve cord expression is in midline precursors, several ventral and intermediate column neuroblasts, and later in neurons but not glia, similar to the known expression of Nkx6 genes in the neural tube. We show genetically that Nk6 is positively regulated, directly or indirectly, by vnd in brain precursors. In vnd mutants, head neuroectoderm Nk6 expression is abolished where it is normally co-expressed with vnd. Conversely, vnd-overexpression leads to ectopic Nk6 expression in the brain. These findings further highlight the importance of interactions between Nk(x)-type genes in regulating their expression.

The Nk-2 box of the Drosophila homeodomain protein, Vnd, contributes to its repression activity in a Groucho-dependent manner

The transcription factor, Vnd, is a dual regulator that specifies ventral neuroblast identity in Drosophila by both repressing and activating target genes. Vnd and its homologues have a conserved amino acid sequence, the Nk-2 box or Nk specific domain, as well a conserved DNA-binding homeodomain and an EhI-type Groucho interaction domain. However, the function of the conserved Nk-2 box has not been fully defined. To explore its function, we deleted the Nk-2 box and compared the regulatory activity of mutant Vnd in transgenic over-expression assays to that of the wild-type protein. We were unable to assign regulatory activity to the Nk-2 box using an over-expression assay, because the mutant protein activated expression of endogenous Vnd, masking a requirement for the Nk-2 box. However, in transgenic rescue assays, Vnd lacking the Nk-2 box repressed ind expression at 30% lower levels than the wild-type protein. Moreover, in transient transfection assays using Gal4 DNA-binding domain-Vnd chimeras, the repression activity of Vnd lacking the Nk-2 box was compromised. Because Vnd represses target gene expression in conjunction with Groucho, we asked whether the Nk-2 box affects Vnds ability to interact with this co-repressor. Vnd lacking the Nk-2 box binds Groucho 30% less efficiently than wild-type Vnd in co-immunoprecipitations. These data suggest that the Nk-2 box contributes to the repression activity of Vnd by stabilizing its interaction with the co-repressor, Groucho.

Conserved properties of the Drosophila homeodomain protein, Ind

Ind-Gsh-type homeodomain proteins are critical to patterning of intermediate domains in the developing CNS; yet, the molecular basis for the activities of these homeodomain proteins is not well understood. Here we identify domains within the Ind protein that are responsible for transcriptional repression, as well as those required for its interaction with the co-repressor, Groucho. To do this, we utilized a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays. We show that Inds candidate Eh1 domain is essential to the embryonic repression activity of this protein, and that Groucho interacts with Ind via this domain. However, when activity is assayed in transient transfection assays using Ind-Gal4 DNA binding domain chimeras to determine domain activity, the repression activity of the Eh1 domain is minimal. This result is similar to previous results on the transcription factors, Vnd and Engrailed. Furthermore, the Eh1 domain is necessary, but not sufficient, for binding to Groucho; the C terminus of Ind, including the homeodomain also affects the interaction with this co-repressor in co-immunoprecipitations. Finally, we show that aspects of the cross-repressive activities of Ind/Gsh2-Ey/Pax6 are evolutionarily conserved. Taken together, these results point to conserved mechanisms used by Gsh/Ind-type homeodomain protein in regulating the expression of target genes.

The Drosophila homeodomain transcription factor, Vnd, associates with a variety of co‐factors, is extensively phosphorylated and forms multiple complexes in embryos

Vnd is a dual transcriptional regulator that is essential for Drosophila dorsal–ventral patterning. Yet, our understanding of the biochemical basis for its regulatory activity is limited. Consistent with Vnd’s ability to repress target expression in embryos, endogenously expressed Vnd physically associates with the co‐repressor, Groucho, in Drosophila Kc167 cells. Vnd exists as a single complex in Kc167 cells, in contrast with embryonic Vnd, which forms multiple high‐molecular‐weight complexes. Unlike its vertebrate homolog, Nkx2.2, full‐length Vnd can bind its target in electrophoretic mobility shift assay, suggesting that co‐factor availability may influence Vnd’s weak regulatory activity in transient transfections. We identify the high mobility group 1‐type protein, D1, and the novel helix–loop–helix protein, Olig, as novel Vnd‐interacting proteins using co‐immunoprecipitation assays. Furthermore, we demonstrate that both D1 and Olig are co‐expressed with Vnd during Drosophila embryogenesis, consistent with a biological basis for this interaction. We also suggest that the phosphorylation state of Vnd influences its ability to interact with co‐factors, because Vnd is extensively phosphorylated in embryos and can be phosphorylated by activated mitogen‐activated protein kinase in vitro. These results highlight the complexities of Vnd‐mediated regulation.

The Drosophila Nkx6 homeodomain protein has both activation and repression domains and can activate target gene expression

Consistent with the common role of Nkx6 family members in specifying motor neuron identity, we show that over-expression of Drosophila Nkx6 results in an increase in the number of Fasiclin II expressing motor neurons in the intersegmental nerve B branch. Our dissection of the regulatory domains of Nkx6 using chimeric cell culture assays revealed the presence of two repression domains and a single activation domain within this transcription factor. As well as its conserved homeodomain, Nkx6 also has a candidate Engrailed homology 1 (Eh1) domain that is conserved amongst all NKx6 family members, through which vertebrate NKx6-type proteins bind the co-repressor, Groucho (Muhr, J., et al., 2001. Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104, 861-73). Paralleling our previous reports that the Eh1 domain of Vnd and Ind are ineffective in Gal4 chimeric assays (Von Ohlen, T., Syu, L.J., Mellerick, D.M., 2007. Conserved properties of the Drosophila homeodomain protein. Ind. Mech. Dev. 124, 925-934; Yu, Z., et al., 2005. Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator. Nucleic Acids Res. 33, 1-12), we found that the Eh1 domain of Nkx6 did not significantly enhance repression in Gal4 chimeric assays. However, when we performed co-immunoprecipitation analyses, we found that Nkx6 can bind Groucho and that binding of Nkx6 to this co-repressor is modulated intra-molecularly. Full length Nkx6 interacted with Groucho poorly, because sequences at the carboxyl terminal of NKx6 interfere with Groucho binding, despite the presence of the Eh1 domain. In contrast, a carboxyl terminal Nkx6 deletion bound Groucho strongly. In keeping with the presence of an activation domain within Nkx6, we also report that Nkx6 can activate reporter expression driven by an Nkx6.1 enhancer that mediates auto-activation in transient transfection assays. The presence of multiple repression domains in Nkx6 supports Nkx6s role as a repressor, potentially using both Groucho-dependent and independent mechanisms. Thus, Nkx6 likely functions as a dual regulator in embryos.

Erratum: Nk6, a novel Drosophila homeobox gene regulated by vnd (Mechanisms of Development (2002) 116 (105-116) PII: S0925477302001557)

Corrigendum to “Nk6, a novel Drosophila homeobox gene regulated by vnd” [Mech. Dev. 116 (2002) 105–116] Jay Uhler, James Garbern, Li Yang, John Kamholz, Dervla M. Mellerick* Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA Karmanos Cancer Institute, Detroit, MI, USA

Corrigendum to “Nk6, a novel Drosophila homeobox gene regulated by vnd”: [Mech. Dev. 116 (2002) 105–116]

Corrigendum to “Nk6, a novel Drosophila homeobox gene regulated by vnd” [Mech. Dev. 116 (2002) 105–116] Jay Uhler, James Garbern, Li Yang, John Kamholz, Dervla M. Mellerick* Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA Karmanos Cancer Institute, Detroit, MI, USA

Corrigendum to “Nk6, a novel Drosophila homeobox gene regulated by vnd”

Corrigendum to “Nk6, a novel Drosophila homeobox gene regulated by vnd” [Mech. Dev. 116 (2002) 105–116] Jay Uhler, James Garbern, Li Yang, John Kamholz, Dervla M. Mellerick* Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA Karmanos Cancer Institute, Detroit, MI, USA