hunchback

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. Using LexA-Hb as bait, cDNAs were isolated representing six different genes. In interaction tests with various unrelated LexA baits, proteins encoded by three of the six cDNAs interacted exclusively with Hb. Among these proteins, the hip76 clone product exhibits the strongest interaction with Hb. Multiple cDNA clones were isolated that span a complete open reading frame (ORF) encoding a 1982-amino acid protein with high sequence similarity to the human autoantigen. dMi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain (Kehle, 1998).

To map the dMi-2-interacting domain in Hb, Hb fragments were generated and tested for dMi-2 interaction in yeast two-hybrid assays. dMi-2 interacts very strongly with sequences overlapping the D domain, a stretch of amino acids that is conserved between Hb proteins of different insect species. Mutations in the D box cause extensive derepression of HOX genes of the Bithorax complex (BXC). Both D box alleles are premature termination codons, suggesting that the D domain and its COOH-terminal flanking sequences are critical for repression of BXC genes. The interaction tests show that this protein portion of Hb interacts with dMi-2. In vitro binding assays with bacterially expressed dMi-2 and Hb proteins confirm that these proteins bind directly to each other. Thus, dMi-2 binds to a portion of Hb that appears to be critical for repression of BXC genes (Kehle, 1998).

dMi-2 homozygotes survive until the first or second larval instar. Mutant embryos and larvae show no obvious mutant phenotypes. Specifically, expression of BXC genes such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B) is completely normal in these mutant embryos. This normal expression may be due to maternally deposited dMi-2 RNAs or proteins that persist through subsequent development. Consistent with this, all early embryos from a dMi-2 deletion stock (including those lacking the gene) show the same high levels of dMi-2 RNA. An attempt was made to generate embryos from mutant dMi-2 germ cells. However, germ cells that are mutant for any of the seven tested dMi-2 alleles fail to develop. This failure can be rescued by a dMi-2 transgene, demonstrating that dMi-2 is essential for the development of germ cells (Kehle, 1998).

An attempt was made to detect a genetic interaction between dMi-2 and hb. hb9Q mutants (carrying a premature stop codon upstream of the first finger domain) show only slight anterior derepression of Ubx in embryos because of perdurance of maternal hb products. hb9K57 mutants (carrying a D box lesion) show more extensive anterior derepression of Ubx; this mutant protein is thought to have dominant-negative effects on the persisting maternal wild-type product. dMi-24;hb9K57 double mutants show much more extensive derepression of Ubx than hb9K57 mutants. Similarly, dMi-24;hb9Q double mutants show more extensive derepression than hb9Q mutants alone. These results demonstrate a synergy between hb and dMi-2 that is consistent with the finding that dMi-2 binds to Hb. Furthermore, it provides strong evidence that dMi-2 functions in the repression of BXC genes (Kehle, 1998).

dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that dMi-2 functions in PcG repression (Kehle, 1998).

Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis suggests that dMi-2 is required for the survival of somatic cells. Do dMi-2 mutations exhibit gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of Ubx, larvae transheterozygous for both Pc and dMi-2 mutations show more extensive derepression. Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in dMi-2/Pc transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in the double mutant. These results are further evidence that dMi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

Within the basal transcription factor complex TFIID, two specific targets, TAFII110 and TAFII60, serve as coactivators to mediate transcriptional activation by BCD and HB. A quadruple complex containing TATA binding protein (TBP), TAFII250, TAFII110, and TAFII60 mediate transcriptional synergism by BCD and HB, whereas triple TBP-TAFII complexes lacking one or the other target coactivator fail to support synergistic activation. Deoxyribonuclease I footprint protection experiments reveal that an integral step leading to transcriptional synergism involves the recruitment of TBP-TAFII complexes to the promoter by way of multivalent contacts between activators and selected TAFIIs. Thus, the concerted action of multiple regulators with different coactivators helps to establish the pattern and level of segmentation gene transcription during Drosophila development (Sauer, 1996).

Krüppel can associate with the transcription factors encoded by gap genes knirps and hunchback, affecting Krüppel-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound HB protein or free KNI protein with distinct but different regions of Krüppel results in the formation of DNA-bound transcriptional repressor complexes (Sauer, 1995).

Translational regulation of Hunchback mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3'-untranslated region of HB mRNA (the Nanos response elements or NREs), as well as two trans-acting factors -- Nanos and Pumilio. Pum binds to a pair of 32-nucleotide sequences (named Nanos response elements -- NREs) in the 3'-UTR of maternal HB mRNA in order to repress HB translation in the posterior of the embryo. This translational repression is essential for normal abdominal segmentation. The RNA-binding domain of Pum is structurally similar to that of another translational regulator, FBF (fem-3 mRNA-binding factor) found in C. elegans (Zhang, 1997). The minimal RNA-binding domain of each protein consists of eight imperfect repeats plus flanking residues. These structural similarities define a conserved 'Puf' motif (Pum and FBF) that is found in proteins from diverse organisms from yeast to humans. However, the RNA partner of no other Puf domain protein has been identified, nor is it clear whether other Puf proteins regulate translation or some other aspect of RNA metabolism. Thus, Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report protein-protein and protein-RNA interaction assays in yeast and in vitro were used to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE. Mutant forms of the NRE, Nos, and Pum that do not regulate HB mRNA normally in embryos do not assemble normally into a ternary complex. In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain. These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos. Taken together, these findings suggest models for how Nos and Pum collaboratively target HB mRNA. More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation (Sonoda, 1999).

In one model, Nos simultaneously makes specific contacts with Pum and nucleotides 17-20 of the NRE. On their own, neither the Nos-Pum nor the Nos-NRE contacts are strong enough to recruit Nos to HB mRNA (at least in the presence of competitor proteins and RNAs), because binary complexes with Nos are not detectable. In another model, unbound Pum cannot interact with Nos, but binding to the NRE induces a conformational change in Pum, which subsequently recruits Nos via protein-protein contacts. In this model, nucleotides 17-20 of the NRE interact with Pum to induce the conformational change without affecting its affinity for the RNA, and nonspecific interactions between Nos and other portions of the RNA help stabilize the complex. Either model is consistent with the nonspecific RNA-binding activity reported for the carboxy-terminal portion of Nos in vitro and the RNA-Nos cross-link found in this study. Further structural and biochemical experiments will be required to distinguish between these (or alternative) models (Sonoda, 1999 and references therein).

The mechanism by which the ternary complex blocks translation is not yet clear. mRNAs subject to Nos- and Pum-dependent repression are deadenylated in vivo. In addition, Nos and Pum have been shown to regulate internal ribosome entry site (IRES)-dependent translation in imaginal disc cells, suggesting that their regulatory target lies downstream of cap recognition and scanning. It is assumed that some surface of the ternary complex, formed jointly by Nos and Pum, targets a component of the polyadenylation or translation machinery. This surface appears to be altered in the Pum680 mutant protein, which binds the NRE normally but is defective in regulating HB translation in the embryo. The Pum680 mutant recruits Nos into a ternary complex normally and thus apparently is defective in a subsequent step of the repression reaction. The RNA-binding domain of Pum therefore appears to have at least three different functions in regulating HB: recognizing the NRE, recruiting Nos, and acting as a corepressor (with Nos) to block translation (Sonoda, 1999 and references therein).

In the experiments reported in this study, focus was placed on discrete regions of both Nos (the carboxy-terminal 97 amino acids) and Pum (the minimal RNA-binding domain), which play an essential role in formation of the ternary complex. However, other regions of Nos are known to be required for its function in repressing translation in the embryo. In addition, residues elsewhere in Pum play an unknown role in augmenting the intrinsic translational repression activity of the RNA-binding domain. Thus, the ternary complex formed by the 157-kD, full-length Pum protein may be stabilized by auxillary protein-protein or protein-RNA interactions in addition to those that mediate recruitment of the carboxy-terminal domain of Nos by the RNA-binding (or Puf domain) of Pum. The results suggest that Puf domain proteins generally may act by recruiting cofactors to specific RNA binding sites. Cofactor specificity may be mediated, at least in part, by the eighth repeat of the Puf domain. Although Puf domain proteins have been described in organisms from yeast to humans, for only one protein other than Drosophila Pum, C. elegans FBF, is the relevant RNA regulatory target known. FBF regulates the sperm/oocyte switch in the hermaphrodite germ line by governing the translation of fem-3 mRNA (Zhang, 1997). The FBF RNA-binding domain interacts with one of the C. elegans Nos homologs (Kraemer, 1999). Further experiments will be required to determine whether the Pum/fly Nos complex and the FBF/worm Nos complex function in a similar manner (Sonoda, 1999 and references therein).

Selective dimerization of Hunchback and Ikaros

The C2H2 zinc finger is the most prevalent protein motif in the mammalian proteome. Two C2H2 fingers in Ikaros are dedicated to homotypic interactions between family members. These fingers comprise a bona fide dimerization domain. Dimerization is highly selective, however, since homologous domains from the TRPS-1 and Drosophila Hunchback proteins support homodimerization, but not heterodimerization with Ikaros. Ikaros-Hunchback selectivity is determined by 11 residues concentrated within the alpha-helical regions typically involved in base recognition. Preferential homodimerization of one chimeric protein predicts a parallel dimer interface and establishes the feasibility of creating novel dimer specificities. These results demonstrate that the C2H2 motif provides a versatile platform for both sequence-specific protein-nucleic acid interactions and highly specific dimerization (McCarty, 2003).

The consensus sequence for C2H2 zinc fingers is (F/Y)-X-C-X_2-5-C-X₃-(F/Y)-X₅-psi-X₂-H-X_3-5-H, where X is any amino acid and psi is a hydrophobic residue. In addition to the cysteines and histidines that coordinate zinc, C2H2 fingers contain conserved hydrophobic residues that pack in the hydrophobic core. These conserved amino acids lead to the formation of the characteristic structure comprised of a two-stranded antiparallel ß sheet and an alpha helix. Structures of several C2H2 domains bound to DNA have been solved by X-ray crystallography and NMR. The structures reveal that nucleotide base contacts are mediated primarily by residues near the N-terminal half of the alpha helix. The four residues most commonly involved in specific base recognition are at positions -1, 2, 3, and 6, relative to the beginnning of the alpha helix (McCarty, 2003).

Although most C2H2 fingers appear to contribute to protein-DNA and protein-RNA interactions, C2H2 fingers have also been implicated in protein-protein interactions. However, structural information has been obtained only for domains in which the contribution of the C2H2 finger to the protein-protein interaction is indirect. For example, dimerization of the RAG1 recombinase is mediated by a domain containing a RING finger and a C2H2 zinc finger. In this domain, the C2H2 finger helps form a stable scaffold upon which the dimer interface is formed, but the finger does not directly participate in dimerization (McCarty, 2003).

Ikaros is a protein expressed in hematopoietic cells that has been implicated in gene silencing and activation. Four C2H2 fingers near the N terminus of Ikaros are involved in sequence-specific DNA binding. The C terminus contains two additional C2H2 zinc fingers that play no apparent role in the protein-DNA interaction. Rather, previous studies using yeast two-hybrid screens and coimmunoprecipitation assays have demonstrated that they are essential for self-interactions and for interactions with the corresponding zinc fingers of other Ikaros family members. A protein fragment spanning the two zinc fingers was sufficient for the interaction, which was disrupted by mutations in the zinc-coordinating cysteines and histidines and by zinc displacement (McCarty, 2003).

Although the C-terminal fingers of Ikaros do not directly contribute to protein-DNA interactions, they are critical for high-affinity DNA binding, which usually involves the recognition of tandem binding sites by two subunits of an Ikaros complex. Homotypic interactions mediated by the C-terminal fingers are also necessary for the targeting of Ikaros to pericentromeric heterochromatin, which has been hypothesized to be important for the pericentromeric recruitment and heritable silencing of Ikaros target genes. Although the C-terminal fingers are involved in protein-protein interactions, rather than protein-DNA interactions, both fingers match perfectly the C2H2 consensus sequence, with the exception of a single missing F/Y in the second finger. The absence of this F/Y does not explain its unusual function, however, since a hydrophobic residue is missing at this position in several DNA binding zinc fingers, including the first and third fingers of the DNA binding domain of Ikaros (McCarty, 2003).

The zinc finger organization found in mammalian Ikaros proteins also exists within the Drosophila gap segmentation protein Hunchback. The C-terminal fingers of Ikaros and Hunchback exhibit considerable sequence homology, suggesting that the Hunchback fingers may support dimer formation. To examine this possibility, the Hunchback C-terminal fingers were substituted for the Ikaros dimerization zinc finger (DZF) domain in the context of both the epitope-tagged protein (f-HbDZF) and untagged IK I (Hb I). Hb I efficiently coimmunoprecipitates with f-HbDZF, confirming that Hunchback C-terminal fingers contain a functional DZF domain. Interestingly, Hb I did not coimmunoprecipitate with f-IkDZF, and IK I did not coimmunoprecipitate with f-HbDZF. These results demonstrate that DZF-mediated dimerization is selective. This surprising degree of selectivity was equally apparent in chemical crosslinking experiments (McCarty, 2003).

To identify the amino acid determinants of selective dimerization, the Ikaros and Hunchback DZF sequences were first compared. This comparison revealed striking similarities and differences. Within the 60 amino acid region, 23 of the amino acids (38%) are identical, consistent with the conserved functions of the domains. The atypical length of the spacer between the final two histidines (5 residues) is also conserved. Despite the high degree of identity, five positions contain charge reversals, ten positions vary between charged and nonpolar residues, and seven positions vary between polar and nonpolar residues. These three categories represent 37% of the DZF amino acids, making it difficult to predict which residues are responsible for dimerization selectivity. Comparison of the Ikaros and TRPS 1 DZF sequences, or the Hunchback and TRPS 1 sequences, reveals similar degrees of identity and divergence (McCarty, 2003).

Because of the high degree of divergence, a systematic approach was necessary to identify the determinants of selective dimerization. As a starting point, two series of Ikaros-Hunchback DZF chimeras were generated in the context of untagged IK. These chimeras were expressed in HEK 293 cells along with either f-HbDZF or f-IkDZF. Protein-protein interactions were monitored using a coimmunoprecipitation assay (McCarty, 2003).

The first series of chimeras contains decreasing amounts of the Hunchback DZF N terminus fused to increasing amounts of the Ikaros DZF C terminus (Hb-IK1 to Hb-IK6). An efficient interaction with f-HbDZF was retained when Ikaros amino acids 53-64 were included in the chimeric protein, but not when amino acids 47-64 were included. These results suggest that at least 1 residue between amino acids 47 and 52 is required for selective homodimerization of the Hunchback DZF, but that amino acids 53-64 are not involved in selectivity (McCarty, 2003).

The second series of chimeric proteins was used to define the amino acids at the N terminus of the Hunchback DZF that are required for selective dimerization. This series contains decreasing amounts of the Ikaros DZF N terminus fused to increasing amounts of the Hunchback DZF C terminus (Ik-Hb1 to Ik-Hb6. An efficient interaction with the f-HbDZF protein was observed when Ikaros amino acids 1-14 were included in the chimeric protein, but the interaction was lost when Ikaros amino acids 1-27 were included. These results demonstrate that at least 1 residue between amino acids 15 and 27 is required for selectivity and that amino acids 1-14 are not involved. Thus, the residues involved in selective dimerization of the Hunchback DZF are located between amino acids 15 and 52 (McCarty, 2003).

The two sets of chimeric proteins were then used to define the region required for dimerization with the Ikaros DZF. Using the Hb-Ik series, an efficient interaction with f-IkDZF was observed when the chimeric protein included Hunchback amino acids 1-27. However, an interaction was not observed when Hunchback amino acids 1-31 were included. Using the Ik-Hb series, an efficient interaction with the f-IkDZF protein was observed when the chimeric protein included Hunchback amino acids 52-60. Inefficient coimmunoprecipitation was observed when the chimeric protein included Hunchback amino acids 34-60, and no coimmunoprecipitation was observed when Hunchback amino acids 32-60 were included. These results suggest that the residues contributing to selective dimerization of the Ikaros DZF are located between amino acids 28 and 51 (McCarty, 2003).

Using a homology model of the Ikaros DZF, it is noted that 7 of the 8 residues identified in this manuscript as important for selective dimerization cluster on the surface of the model. These patches are likely to represent the dimer interface. The clusters correspond primarily to the alpha helices of both zinc finger domains. Within the first finger, the clustering of the 3 critical residues, L17, M21, and C28, was expected on the basis of the 7 residue spacing between M21 and C28 on the alpha helix, and the 4 residue spacing between M21 and L17. Because the orientation of the alpha helix is constrained by the conserved hydrophobic residue, Y22, which packs in the hydrophobic core, there is a high probability that these critical residues will be exposed in the DZF structure (McCarty, 2003).

In the second finger, the 4 residues that are most important for selective dimerization, R47, Y48, E49, and F50, are adjacent to one another on the alpha helix. A hydrophobic residue that packs in the core is expected at the position occupied by F50. Therefore, this residue is almost certainly at the core and does not directly contribute to the dimer interface. Instead, F50 is likely to make an important contribution to the structure of the finger. Interestingly, this residue cannot be replaced by the leucine found at the same position in the Hunchback DZF. This observation suggests that the structures of the hydrophobic cores of the Ikaros and Hunchback DZFs differ, which may be important for selectivity (McCarty, 2003).

Residues R47, Y48, and E49 are likely to be important constituents of the dimer interface. Because the hydrophobic cores of C2H2 motifs are highly compact, all three of these residues are likely to be exposed. However, the precise orientations of the amino acid side chains cannot be determined in the absence of an experimentally defined structure. The presence of arginine, tyrosine, and glutamate residues at the Ikaros dimerization interface (R47, Y48, and E49) suggests that a combination of salt bridges, hydrogen bonds, and hydrophobic interactions stabilize the dimer. Equivalent positions of the Hunchback DZF sequence contain hydrophobic residues, suggesting that stabilization of the Hunchback dimerization interface may be more dependent on van der Waals interactions (McCarty, 2003).

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.