BIOLOGICAL OVERVIEW

Many genes with complex developmental regulation contain multiple enhancers, the binding sites for transcription factors that function at quite a distance from gene coding sequences. It is thought that higher eukaryotes possess factors that facilitate remote enhancer-promoter interactions. Such enhancer-facilitators may be envisioned as helping to form chromatin structures that bring enhancers and promoters closer together; they are different from enhancer-binding activators, coactivators, and basal factors in that they do not participate directly in the activation reaction. Enhancers can interact with proximal promoters from distances of thousands of base pairs. The function of enhancers is disrupted by the Drosophila protein Suppressor of Hairy-wing (Su[Hw]). Su (Hw) binds a DNA sequence in the gypsy retrotransposon and prevents distal enhancers with intervening gypsy insertions from activating target genes without affecting promoter-proximal enhancers. Several observations indicate that su(Hw) does not affect enhancer-binding activators. Instead, su(Hw) may interfere with factors that structurally facilitate interactions between an enhancer and promoter.

To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped (sd), mastermind,(mam) and a previously unknown gene, Chip, have been isolated. A TEA DNA-binding domain in the Scalloped protein binds the cut wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

The data suggest that sd and mam encode enhancer-binding factors and that Chip may encode an enhancer-facilitator. Both sd and mam mutants display stronger genetic interactions with wing margin enhancer deletions than with gypsy insertions in cut (gypsy insertions block enhancers with the help of su(Hw), a transcription factor that binds the gypsy retrovirus). Chip is also needed for wing margin enhancer activity but appears to play a unique role. Chip is normally required for wing margin enhancer function because Chip mutations enhance the cut wing phenotype of cut mutants. However, in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. In a Chip heterozygote (with the wild-type chromosome able to carry out Chip mediated activation of cut), a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) protein bound to a gypsy insertion in one cut allele acts in a transvection-like manner (one chromosome influencing the activity of the second) to block the wing margin enhancer in the wild-type cut allele on the other chromosome. The simplest interpretation is that Chip protein facilitates enhancer-promoter communication and su(Hw) on one chromosome interferes with Chip mediated enhancer-promoter communication on both chromosomes (Morcillo, 1996 and 1997).

Chip is a LIM protein interactor, as are Chip vertebrate homologs. Chip interacts directly with the LIM domains of Apterous. Chip maternal mutations play a role in segmentation, and evidence supports a role for Chip in regulating the gap gene giant, and possibly the pair-rule gene even-skipped. Moreover, Chip regulates expression of cut and Ultrabithorax during imaginal disc development; these genes are not known to be regulated by LIM domain proteins. Although the role(s) of LIM domain proteins in early Drosophila development is currently unknown, it is possible that LIM domain proteins play broader roles in development than appreciated previously, and that several unknown LIM domain proteins are required for segmentation and imaginal disc development. Another possibile explanation for the broad functions of Chip is that it may interact with other proteins without LIM domains. The two mouse Chip homologs, Nli/Lbd1/Clim-2 and Clim-1 interact directly with P-Otx, a homeodomain protein that lacks LIM domains (Morcillo, 1997).

Does Chip play an novel role in enhancer functions different from that played by transcriptional co-activators? Transcriptional co-activators may be thought of as proteins that serve as a bridge interacting with transcription factors and activating the transcriptional apparatus. Chip and its vertebrate homologs appear to regulate interactions between different transcriptional activator proteins and may function at enhancers to bring together diverse transcriptional factors and form higher order activation complexes; in some cases, to block formation of such complexes. Specific antagonism between Chip and suppressor of Hairy wing suggests a role for Chip in enhancer-promoter communications. The diverse roles suggested for Chip suggest a distinction between Chip and roles for transcriptional co-activators whose targets are thought to be the transcriptional apparatus (Morcillo, 1997).

Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers (Torigoi, 2000).

Full-length Chip interacts with the HD proteins Bicoid (Bcd) and Ftz, and with a fragment of the Su(Hw) insulator protein. The HD protein Otd binds almost as efficiently as does Bcd and Ftz to Chip, but the Eve HD protein binds poorly, a result possibly attributable to improper folding of the in vitro-translated protein. The domains of Chip involved in homotypic and heterotypic interactions include the LIM interaction domain (LID) and the self-interaction domain (SID). Deletion of the LID reduces interaction with Apterous. That deletion, however, has no effect on interaction with Bcd, Ftz, Su(Hw)DeltaCTD, or Chip. In contrast, two other deletion mutants, ChipDelta404-465 and ChipDelta441-454, reduce binding to Bcd, Ftz, Su(Hw)DeltaCTD, and Chip but have little effect on binding to Apterous. On the basis of this and additional deletion mutants, Chip residues 439-456 are identified as the region that interacts with the HD proteins, Su(Hw), and with Chip itself. This region is termed the other interaction domain (OID) (Torigoi, 2000).

Previous studies have suggested that the SID is sufficient for self-interaction of Chip, but Chip self-interaction is reduced by deletions affecting the OID (ChipDelta404-465 and ChipDelta441-454) but is unaffected by a deletion that removes much of the SID (ChipDelta294-381). An isolated SID fragment (ChipDelta404-519) interacts with itself but does not interact well with intact Chip, whereas a Chip fragment lacking the SID (ChipDelta2-381) interacts both with itself and with intact Chip. Experiments that show interactions between the SID and intact Chip were performed by translating the two interaction partners together in vitro. Evidently, cotranslation permits an interaction not seen by affinity chromatography. It is concluded that Chip interacts with itself through both the SID and the OID (Torigoi, 2000).

The domains of Bcd and Su(Hw) that interact with Chip were mapped to determine if the OID recognizes a common motif in its diverse interaction partners. The N-terminal half of Bcd (residues 1-255) contains the HD and everything needed to rescue bcd mutants in vivo. The N-terminal half of Bcd interacts with Gst-Chip, whereas the C-terminal half (residues 246-489) does not. Smaller Bcd fragments containing the HD (residues 1-190, 1-166 or 57-255) bind more weakly than does the 1-255 fragment, and a fragment (residues 57-166) consisting mostly of the HD (residues 92-151) does not bind. Thus, residues on both sides of the HD are required for strong binding. Similar results were obtained with the Otd HD protein. The region of Su(Hw) that contains 12 zinc fingers (residues 204-672) interacts with Gst-Chip, whereas the N-terminal region (residues 1-190) and the C-terminal region (residues 706-944) do not. Mutation of any one of the 12 zinc fingers does not significantly affect binding to Chip. The regions of Bcd, Su(Hw), and Chip that interact with the Chip OID are not homologous at the primary sequence level (Torigoi, 2000).

The interactions between Chip and HD proteins in vitro raise the question of whether Chip affects the activities of HD proteins in vivo. The effect of Chip on Bcd activity in embryos was tested because both Chip and Bcd are provided maternally and do not regulate each other's expression. Thus, any effect of Chip on Bcd is likely to be direct. The design of the experiment that shows that in embryos reducing Chip activity decreases the activity of a partially defective Bcd protein, was guided by the following considerations. To demonstrate a helping effect of Chip on Bcd activity, maternal Chip could not be simply eliminated because that manipulation results in a more severe segmentation defect than does elimination of Bcd itself. Nor could the dosage of maternal Chip be halved because that change has no effect, even if the maternal Bcd level is also reduced by one-half. Moreover, zygotic Bcd makes no contribution to segmentation; heretofore, no effect has been seen on segmentation by changing the level or nature of zygotically expressed Chip. To detect an effect of Chip on Bcd activity, therefore, the activities of both Bcd and Chip were reduced to less than that provided by a single maternal dose of each. This was accomplished by producing doubly mutant mothers: these mothers were homozygous for the bcd^E3 allele, which encodes a mutant with reduced DNA-binding activity, and were also heterozygous for the Chip^g96.1 allele. This latter mutant allele encodes the SID fragment, which acts as a dominant negative, inhibiting, but not eliminating, maternal Chip activity. It was deduced that the SID fragment inhibits maternal Chip activity from the observations that Chip^g96.1/Chip^g96.1 embryos produced by Chip^g96.1/+ mothers die before reaching the larval stage (some display a mild segmentation defect), whereas all Chip^-/Chip^- embryos produced by Chip^-/+ mothers segment normally and die as larvae. It was further deduced that at least one maternal and two zygotic doses of the SID fragment are required to cause embryonic lethality from the fact that Chip^g96.1/+ embryos from Chip^g96.1/+ mothers segment normally and survive to adulthood. Presumably the SID fragment, produced in this experiment both maternally and zygotically, forms nonfunctional multimers with maternal wild-type Chip. On average, embryos from Chip^g96.1/+; bcd^E3/bcd^E3 mothers (and wild-type fathers) produce nearly one segment less than do embryos from bcd^E3/bcd^E3 mothers (Torigoi, 2000).

These results suggest that Chip increases interactions between Bcd molecules. Thus, in yeast with nonsaturating levels of Bcd, Chip increases activation by Bcd from two strong binding sites separated by a weak site or by a nonbinding spacer, but not from one or three contiguous strong sites. Moreover, Chip does not increase activation above levels that are achieved with high concentrations of Bcd itself. Bcd binds DNA cooperatively, mediated by interactions of regions overlapping those that interact with Chip, and it is suggested that Chip interacts with Bcd to amplify that cooperativity. It is unlikely that Chip itself is a transcriptional activator. Previous experiments have shown that Chip does not activate when tethered upstream of yeast promoters but it can induce activation by recruiting an activation domain fused to LIM domains (Torigoi, 2000).

The idea that Chip increases interactions between certain other proteins agrees with all previous observations on Chip and its homologs. In transient transfection experiments with mammalian cells, Chip homologs increased transcriptional activation by the combination of the P-Otx HD and the Lhx3 LIM-HD proteins from a promoter containing a single binding site for each molecule. The Chip homologs have little effect with P-Otx or Lhx3 alone, indicating that they aid P-Otx-Lhx3 interactions. Furthermore, the nuclear LIM interactor (Nli) homolog of Chip aids formation of different LIM-HD protein dimers in vitro, an effect requiring the Nli SID. Finally, an Apterous-Chip fusion protein, in which the LIM domains of Apterous are replaced by the Chip SID, can replace wild-type Apterous in Drosophila wings, suggesting that Chip aids formation of Apterous dimers in vivo (Torigoi, 2000 and references therein).

Chip potentiates Bcd activity in the Drosophila embryo when the Bcd activity is low. This effect is consistent with previous studies on the expression of segmentation genes in embryos lacking maternal Chip activity. Embryos contain a gradient of Bcd protein, with a high concentration at the anterior end and a low concentration at the posterior end. Loss of maternal Chip strongly reduces all seven blastoderm stripes of Eve protein produced by the eve pair-rule gene. Many, if not all of these stripes are also regulated by Bcd, even though most occur in regions with low to intermediate Bcd concentrations. The eve stripes are activated by several remote enhancers located ~1.5-9 kb from the promoter, and Bcd-binding sites are critical for activation by at least the stripe 2 enhancer. It is likely, therefore, that Chip increases eve expression at least in part by increasing binding of Bcd to the enhancers. Accumulation of the Hb protein is not substantially affected by loss of maternal Chip even though hb expression is dependent on Bcd and several Bcd-binding sites just upstream of the promoter. This lack of an effect of Chip is not unexpected, however, because hb is expressed in the anterior end where the Bcd concentration is the highest (Torigoi, 2000 and references therein).

It is suggested that Chip plays two roles in the regulation of gene expression: (1) Chip is likely to aid binding of proteins to enhancers, and (2) Chip is also likely to function between enhancers and promoters to support enhancer-promoter communication. The in vitro interaction between Chip and the Su(Hw) insulator protein shown here is consistent with the notion that Su(Hw) is directly antagonistic to Chip activity as previously demonstrated genetically at the cut locus. It remains to be seen how, if these speculations are correct, Chip facilitates enhancer-promoter communication and how that communication is disrupted by Su(Hw). It is believed that Su(Hw) blocks activation not by reducing the binding of proteins to enhancers, but rather by hindering enhancer-promoter communication. For instance, an enhancer blocked in its interaction with one promoter by Su(Hw) can nevertheless activate a second promoter located on the opposite side of the enhancer from Su(Hw). Thus, although Su(Hw) is antagonistic to Chip, it is unlikely to affect binding of proteins to enhancers. It is also unlikely that Chip functions merely by preventing binding of Su(Hw) to gypsy because Chip is also important for the expression of several genes, e.g., cut and eve, in the absence of gypsy and Su(Hw) (Torigoi, 2000).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 575

Structural Domains

The protein encoded by Chip is homologous to Nli/Lbd1/Clm-2 and frog Xlbd1 vertebrate proteins that bind to the LIM domains of nuclear proteins. Chip residues 205 to 577 display 58% identity with the mouse Nli/Lbd1/Clm-2 protein. All of these proteins have a potential nuclear localization signal. There are no yeast homologs. The major difference between Chip and the vertebrate homologs is that Chip has a proline-rich amino-terminal domain of about 200 amino acids (Morcillo, 1997).

date revised: 12 January 98

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.