Antennapedia

The Antennapedia and Abdominal-B homeodomains bind to TFIIEß, but the Even-skipped homeodomain does not. Using a two-hybrid assay performed in cultured cells, it can be shown that the interaction occurs in vivo. The Abdominal-B homeodomain is shown to activate transcription in vitro, and this activation can be blocked with anti-TFIIEß antibody without affecting basal transcription levels. How can an interaction between TFIIEß and the homeodomain contribute to transcriptionsal activation? At a biochemical level. TFIIE promotes the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II by TFIIH, but also inhibits a helicase activity shown by TFIIH that may be required to unwind the DNA prior to transcription initiation. Since the phosphorylated RNA polymerase II is the form of the enzyme that actively elongates transcripts, the ability of a homeodomain to attract TFIIE to the initiation complex would serve to stimulate transcription by enhancing the kinase activity of TFIIH resulting in a completely phosphorylated CTD. Alternatively, since TFIIE inhibits the helicase activity of TFIIH, it has been proposed that TFIIE might be removed from the complex following the CTD phosphorylation, and this be facilitated by interaction with a homeodomain (Zhu, 1996 and references).

The two Drosophila homeotic proteins encoded by Antennapedia and Sex combs reduced determine cell fates in the epidermis and internal tissues of the posterior head and thorax. Genes encoding chimeric ANTP/SCR proteins were introduced into flies and their effects on morphology and target gene regulation observed. The N-terminus of the homeodomain appears to be critical for determining the specific effects of these homeotic proteins in vivo, but other parts of the proteins have some influence as well. The N-terminal part of the homeodomain has been observed, in crystal structures and in NMR studies in solution, to contact the minor groove of the DNA. The different effects of Antennapedia and Sex combs reduced proteins in vivo may depend on differences in DNA binding, protein-protein interactions, or both (Zeng, 1993).

The secondary structure of an N-terminally elongated Antennapedia homeodomain (HD) polypeptide containing residues -14 to 67 (where residues 1-60 constitute the HD) has been determined by NMR in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met- (YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm (consisting of residues -14 to 6) is flexibly disordered, and the well-defined part of the HD structure (residues 7-59) is indistinguishable from that of the shorter ANTP HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter ANTP HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the ANTP protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the ANTP HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact ANTP protein (Qian, 1992).

The Ultrabithorax (UBX), abdominal-A (ABD-A), and ANTP homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress, whereas ANTP activates P1. Either of two regions of P1 can confer this pattern of differential regulation. One of the regions lies downstream and contains homeoprotein-binding sites flanking a 37-bp region called BetBS. ANTP protein activates transcription through the binding sites, whereas UBX and ABD-A both activate transcription through BetBS and use the flanking binding sites to prevent this effect. Thus, homeoproteins can use the same regulatory element but in very different ways. Chimeric UBX-ANTP proteins and UBX deletion derivatives demonstrate that functional specificity in P1 regulation is dictated mainly by sequences outside the homeodomain, with important determinants in the N-terminal region of the proteins (Saffman, 1994).

The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. The DNA sequence preferences were compared of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity (Ekker, 1994).

The Antennapedia homeodomain differs at only five amino acid positions from that of Sex combs reduced protein. In a chimeric Antp-Scr proteins expressed ectopically in Drosophila, the functional specificity of the ANTP protein is determined by the four specific amino acids located in the flexible N-terminal arm of the homeodomain. The three-dimensional structure of the ANTP homeodomain-DNA complex shows that this N-terminal arm is located in the minor groove of the DNA, suggesting that the functional specificity is determined either by slight differences in DNA binding and/or by selective interactions with other transcription factor(s) (Furukubo-Tokunaga, 1993).

One example of the role of Casein kinase II in modification of a Drosophila protein is found in the interaction of CkII with Antennapedia. The in vivo activity of this HOX protein is modified by phosphorylation due to CkII. Antp has four putative CkII target sites. Sites 1 and 2 are found in the amino-terminal portion of the protein, whereas sites 3 and 4 are clustered close to the homeodomain in the C-terminal tail. Antp with alanine substitutions at its CkII target sites produces altered thoracic and abdominal development. Ubiquitous expression of Antp in flies produces an inhibition of head involution, the elimination of dorsal head structures, a transformation of T1 into a second thoracic segment (T2), and the appearance of one to two partial T2 denticle belts in the head segment. Embryos that express Antp with altered CkII target sites (alanine replacing serine or threonine) exhibit additional phenotypes including an absence of Keilin's organs, shortened denticle belts, and a failure of germ-band retraction. Embryos that express altered Antp show a disorganized CNS with irregularly spaced or fused horizonal commissures and gaps in the longitudinal commissures. CkII sites 1 and 4 appear to be the most important in terms of the altered phenotypes produced (Jaffe, 1997).

The novel functions that result from mutationally removing CkII sites suggest that altered Antp is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of Antp that contains acidic amino acid substitutions at its CkII target sites is greatly reduced, mimicking a constitutively phosphorylated Antp protein. This hypoactive form of Antp, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. These results suggest that phosphorylation of Antp by CkII is important for preventing inappropriate activities of this homeotic protein during embryogenesis. The information provided however does not address the mechanism by which phosphorylation alters Antp's properties. Thus phosphorylation appears to modulate Antp's properties, restricting its activity to an appropriate level (Jaffe, 1997).

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