Transcription factor TFIIEβ interacts with two exposed positions in helix 2 of the Antennapedia homeodomain to control homeotic function in Drosophila

Abstract

Homeoproteins contain the conserved homeodomain (HD) and have an important role determining embryo body plan during development. HDs increase their DNA-binding specificity by interacting with additional cofactors outlining a Hox interactome with a multiplicity of protein-protein interactions. In Drosophila, the first link of functional contact with a general transcription factor (GTF) was found between Antennapedia (Antp) and BIP2 (TFIID complex). Hox proteins also interact with other components of Pol II machinery such as the subunit Med19 from Mediator (MED) complex, TFIIEβ and transcription-pausing factor M1BP. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but the precise molecular mechanism remains unclear. In this paper, we focused on the Antp-TFIIEβ protein-protein interface to establish the specific contacts as well as its functional role. Using Bimolecular Fluorescence Complementation (BiFC) in cell culture and in vivo we found that TFIIEβ interacts with Antp through the HD independently of the YPWM motif and the direct physical interaction is at helix 2, specifically aminoacidic positions I32 and H36 of Antp. We also found, through ectopic assays, that these two positions in helix 2 are crucial for Antp homeotic function in head involution, and thoracic and antenna-to tarsus transformations. Interestingly, overexpression of Antp and TFIIEβ in the antennal disc showed that this interaction is required for the antenna-to-tarsus transformation. In conclusion, interaction of Antp with TFIIEβ is important for the functional specificity of Antennapedia, and amino acids 32 and 36 in Antp HD helix 2 are key for this interaction. Our results open the possibility to more broadly analyze Antp-TFIIEβ interaction on the transcriptional control for the activation and/or repression of target genes in the Hox interactome during Drosophila development.

Dynamic Molecular Evolution of Mammalian Homeobox Genes: Duplication, Loss, Divergence and Gene Conversion Sculpt PRD Class Repertoires

“…2 that most of these residues are on the outer surface of the homeodomain. Since both of these helices have been proposed to mediate protein-protein interactions in some homeodomains, including those of the PRD class (Wilson et al 1995;Simon et al 1997;Zaffran and Frasch 2005;Plaza et al 2008;Altamirano-Torres et al 2018), we propose that this selection has altered ETCHbox protein-protein binding properties. Madissoon et al (2016) found that homeodomain differences are not sufficient to explain the differing transcriptional effects of PRD-like genes, implying that other protein domains also contribute to specificity; we therefore suggest that sites outside of the homeodomain that are under selection also influence specificity.…”

Trimeric complexes of Antp-TBP with TFIIEβ or Exd modulate transcriptional activity

“…2 C and F ). In the same way, either the HD deletion of Antp in AntpΔHD or the HD helix 2 mutant Antp I32A−H36A [ 22 ] reduced the formation of the Antp-TBP-TFIIEβ trimer in a highly significant manner ( E = 0.13 ± 0.09 and E = 0.17 ± 0.10 respectively; Fig. 2 D-F).…”

“…TBP interacts with CDX1 via the HD but not with CDX2, supporting the selective interaction of TBP with homeoproteins [ 34 ]. Like other homeoproteins, Antp interacts with other members of the basal transcription machinery as BIP2/TAF3, Med19, TFIIEβ and M1BP [ 14 , 19 – 22 ], denoting the implication of homeoproteins in the Preinitiation Complex (PIC).…”

“…Several homeoproteins interact with the Med19 subunit of MED complex, TFIIEβ and M1BP, a pausing Pol II factor involved in chromatin remodeling [ 14 , 19 , 20 ]. Antennapedia (Antp) interacts with the Bric-a-brac interacting protein (BIP2/TAF3) through the YPWM motif [ 21 ] and with TFIIEβ, specifically through the 32 and 36 positions of HD helix 2 [ 22 ]. Additionally, Drosophila and mouse Hox proteins form trimeric complexes with Exd-Homothorax (Hth) and MEIS-PBX respectively [ 23 , 24 ].…”

Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila

“…An interaction between the bait and prey reconstitutes a detectable fluorescing molecule (Kerppola, 2008). In flies, BiFC has previously been used to detect interactions between cytoskeletal proteins (Gohl et al, 2010), transcription factors (Hudry et al, 2012;Altamirano-Torres et al, 2018;Bischof et al, 2018), and myofibril proteins (González-Morales et al, 2019b). Here, we present a detailed BiFC protocol to study protein-protein interactions in Drosophila myofibrils.…”