An extradenticle-induced conformational change in a HOX protein overcomes an inhibitory function of the conserved hexapeptide motif.

Abstract

HOX homeoproteins control cell identities during animal development by differentially regulating target genes. The homeoprotein encoded by the extradenticle (exd) gene can selectively modify HOX DNA binding, suggesting that it contributes to HOX specificity in vivo. HOX‐EXD interactions are in part mediated by a conserved stretch of amino acids termed the hexapeptide found in many HOX proteins. Here, we demonstrate that a 20 bp oligonucleotide from the 5′ region of the mouse Hoxb‐1 gene, a homolog of Drosophila labial (lab), is sufficient to direct an expression pattern in Drosophila that is very similar to endogenous lab. In vivo, this expression requires lab and exd and, in vitro, LAB requires EXD to bind this oligonucleotide. In contrast, LAB proteins with mutations in the hexapeptide bind DNA even in the absence of EXD. Moreover, a hexapeptide mutant of LAB has an increased ability to activate transcription in vivo. Partial proteolysis experiments suggest that EXD can induce a conformational change in LAB. These data are consistent with a mechanism whereby the LAB hexapeptide inhibits LAB function by inhibiting DNA binding and that an EXD‐induced conformational change in LAB relieves this inhibition, promoting highly specific interactions with biologically relevant binding sites.

A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax

“…Finally, our study questions whether additional HX-independent modes of PBC interaction exist. It was reported previously that the HX-deficient Lab protein retains Exd interaction potential and in vivo Exd-dependent activity (12). As Lab does not bear a UbdA motif, it supports further flexibility in Hox-PBC interaction.…”

“…5 and 6) and by crystallographic studies that showed that the HX provides most if not all major contacts (7)(8)(9). In contrast, in vivo functional support for a role of the HX in mediating interaction with PBC proteins is still limited, mainly because effects of HX mutations during development have only been assessed for two vertebrate Hox proteins, Hoxa-1 (10) and Hoxb-8 (11), and for three Drosophila proteins, Labial (Lab) (12), Ubx (13), and Abdominal-A (AbdA) (14).…”

“…However, addressing in vertebrates whether phenotypes resulting from HX mutations are consequences of defects in Pbx interaction will require examination of combinations of Hox-1 paralogous and Pbx gene mutations. In Drosophila, mutation of the HX in Lab, the only representative of Hox-1 class genes, results in an hyperactive protein when assayed for its potential to activate transcription through an evolutionarily conserved Hoxb-1 autoregulatory element (12,16). This hyperactivity results from the loss of an inhibitory action of the HX on Lab DNA binding.…”

“…This hyperactivity results from the loss of an inhibitory action of the HX on Lab DNA binding. In this context, it was proposed that HX-mediated recruitment of Extradenticle (Exd) acts to mask the DNA-binding inhibitory activity of the HX motif (12).…”

Selection of distinct Hox–Extradenticle interaction modes fine-tunes Hox protein activity

“…Although other proteins presumably are involved, our current knowledge of Hox protein mode of action stems mainly from the interaction with PBC class of cofactors, Pbx in vertebrates and Extradenticle (Exd) in Drosophila (4-7). Extensive data, including in vitro interaction assays (8,9), the requirement for in vivo Exd-dependent processes (10)(11)(12), and crystallographic studies (13)(14)(15)(16), support the notion that Hox-PBC interaction relies on a short hexapeptide (HX) motif lying upstream of the homeodomain (HD) and shared by most Hox proteins.…”