Gene name - Chip
Cytological map position - 60B1-60B2
Function - Transcription factor
Symbol - Chi
FlyBase ID: FBgn0013764
Genetic map position - 2-106.8
Classification - novel protein homologous to Nli/Lbd1/Clm-2 and Xlbd1
Cellular location - nuclear
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 bcdE3 allele, which encodes a mutant with reduced DNA-binding activity, and were also heterozygous for the Chipg96.1 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 Chipg96.1/Chipg96.1 embryos produced by Chipg96.1/+ 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 Chipg96.1/+ embryos from Chipg96.1/+ 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 Chipg96.1/+; bcdE3/bcdE3 mothers (and wild-type fathers) produce nearly one segment less than do embryos from bcdE3/bcdE3 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).
TCF/LEF factors (see Drosophila Pangolin) are ancient context-dependent enhancer-binding proteins that are activated by β-catenin (see Drosophila Armadillo) following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. This study used a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, this study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer (Fiedler, 2015).
TCF/LEF factors (TCFs) were discovered as context-dependent architectural factors without intrinsic transactivation potential that bind to the T cell receptor α (TCRα) enhancer via their high mobility group (HMG) domain. They facilitate complex assemblies with other nearby enhancer-binding proteins, including the signal-responsive CRE-binding factor (CREB) and the lineage-specific RUNX1 (also called Acute Myeloid Leukemia 1, AML1). Their activity further depends on β-catenin, a transcriptional co-factor activated by Wnt signaling, an ancient signaling pathway that controls animal development and stem cell compartments, and whose dysregulation often causes cancer. The context-dependence of TCFs is also apparent in other systems, for example in the embryonic midgut of Drosophila where dTCF integrates multiple signaling inputs with lineage-specific cues during endoderm induction. The molecular basis for this context-dependence remains unexplained (Fiedler, 2015).
In the absence of signaling, T cell factors (TCFs) are bound by the Groucho/Transducin-like Enhancer-of-split (Groucho/TLE) proteins, a family of co-repressors that silence TCF enhancers by recruiting histone deacetylases (HDACs) and by 'blanketing' them with inactive chromatin. TLEs are displaced from TCFs by β-catenin following Wnt signaling, however this is not achieved by competitive binding but depends on other factors. One of these is Pygopus (Pygo), a conserved nuclear Wnt signaling factor that recruits Armadillo (Drosophila β-catenin) via the Legless/BCL9 adaptor to promote TCF-dependent transcription. Intriguingly, Pygo is largely dispensable in the absence of Groucho, which implicates this protein in alleviating Groucho-dependent repression of Wg targets (Fiedler, 2015).
Pygo has a PHD and an N-terminal asparagine proline phenylalanine (NPF) motif, each essential for development and tissue patterning. Much is known about the PHD finger, which binds to Legless/BCL9 and to histone H3 tail methylated at lysine 4 via opposite surfaces that are connected by allosteric communication. By contrast, the NPF ligand is unknown, but two contrasting models have been proposed for its function (1">Figure 1
This study used a proteomics approach to discover that the NPF ligand is an ancient protein complex composed of Chip/LDB (LIM-domain-binding protein) and single-stranded DNA-binding protein (SSDP), also called SSBP. This complex controls remote Wnt- and Notch-responsive enhancers of homeobox genes in flies (Bronstein, 2011), and remote enhancers of globin and other erythroid genes in mammals, integrating lineage-specific inputs from LIM-homeobox (LHX) proteins and other enhancer-binding proteins. Using nuclear magnetic resonance (NMR) spectroscopy, this study demonstrated that Chip/LDB-SSDP (ChiLS) binds directly and specifically to Pygo NPFs, and also to NPF motifs in Runt-related transcription factors (RUNX) proteins and Osa (Drosophila ARID1), whose relevance is shown by functional analysis of Drosophila midgut enhancers. Furthermore, Groucho was identified as another new ligand of ChiLS by mass spectroscopy. This study thus define the core components of a Wnt enhanceosome assembled at TCF enhancers via Groucho/TLE and RUNX, primed for timely Wnt responses by ChiLS-associated Pygo. The pivotal role of ChiLS in integrating the Wnt enhanceosome provides a molecular explanation for the context-dependence of TCFs (Fiedler, 2015).
The discovery of ChiLS as the NPF ligand of Pygo proteins led to the definition of the core components of a multi-protein complex tethered to TCF enhancers via Groucho/TLE and RUNX, and slated for subsequent Wnt responses by Pygo (see Model of the Wnt enhanceosome). ChiLS also contacts additional enhancer-binding proteins via its LID, including lineage-specific and other signal-responsive factors, thereby integrating multiple position-specific inputs into TCF enhancers, which provides a molecular explanation for the context-dependence of TCF/LEF. This complex will be referred to as the Wnt enhanceosome since it shares fundamental features with the paradigmatic interferon β-responsive enhanceosome (Panne, 2007). Its components are conserved in placozoa, arguably the most primitive animals without axis and tissues with only a handful of signaling pathways including Wnt, Notch and TGF-β/SMAD, suggesting that the Wnt enhanceosome emerged as the ur-module integrating signal-responses (Fiedler, 2015).
Other proteins have been reported to interact with the Pygo N-terminus, but none of these recognize NPF. It is noted that this N-terminus is composed of low-complexity (intrinsically disordered) sequences that are prone to non-specific binding (Fiedler, 2015).
NPF is a versatile endocytosis motif that binds to structurally distinct domains, including eps15 homology (EH) domains in epsin15 homology domain (EHD) protein. Indeed, EHDs were consistently identified in lysate-based pull-downs with triple-NPF baits. EHDs are predominantly cytoplasmic, and do not interact with nuclear Pygo upon co-expression, nor are any of the Drosophila EHDs required for Wg signaling in S2 cells. ChiLS is the first nuclear NPF-binding factor (Fiedler, 2015).
NPF binding to ChiLS appears to depend on the same residues as NPF binding to EHD domains, that is, on the aromatic residue at +2, the invariant P at +1, N (or G) at 0 and NPF-adjacent residues, including negative charges at +3 and +4 (whereby a positive charge at +3 abolishes binding to EHD). Indeed, an intramolecular interaction between the +3 side-chain and that of N predisposes NPF to adopt a type 1 β-turn conformation, which increases its affinity to the EHD pocket, while the -1 residue undergoes an intermolecular interaction with this pocket. ChiLS also shows a preference for small residues at -1 and -2, similarly to N-terminal EHDs although RUNX seems to differ at -1 and -2 from Pygo and MACC1 (F/L A/E/D vs S A, respectively) (Fiedler, 2015).
Groucho/TLE is recruited to TCF via its Q domain, which tetramerizes. Intriguingly, the short segment that links two Q domain dimers into a tetramer is deleted in a dTCF-specific groucho allele that abolishes dTCF binding and Wg responses, suggesting that TCF may normally bind to a Groucho/TLE tetramer (Fiedler, 2015).
Groucho/TLE uses its second domain, the WD40 propeller, to bind to other enhancer-binding proteins on Wnt-responsive enhancers, most notably to the C-terminal WRPY motif of RUNX proteins (common partners of TCFs in Wnt-responsive enhancers). This interaction can occur simultaneously with the WD40-dependent binding to ChiLS, given the tetramer structure of Groucho/TLE. In turn, RUNX uses its DNA-binding Runt domain to interact with HMG domains of TCFs, and to recruit ChiLS. RUNX thus appears to be the keystone of the Wnt enhanceosome since it binds to the enhancer directly while undergoing simultaneous interactions with Groucho/TLE (through its C-terminal WRPY motif), TCF and ChiLS (though its Runt domain) (Fiedler, 2015).
In line with this, Runt has pioneering functions in the early Drosophila embryo, shortly after the onset of zygotic transcription, and in the naïve endoderm as soon as this germlayer is formed, in each case prior to the first Wg signaling events. RUNX paralogs also have pioneer-like functions in specifying cell lineages, that is, definitive hematopoiesis in flies and mammals (Fiedler, 2015).
The model predicts that ChiLS, once tethered to the enhanceosome core complex, recruits Pygo via NPF to prime the enhancer for Wnt responses (see Model of the Wnt enhanceosome). Given the dimer-tetramer architecture of ChiLS, its binding to Pygo can occur simultaneously to its NPF-dependent binding to RUNX. In turn, tethering Pygo to the Wnt enhanceosome may require Pygo's binding to methylated histone H3 tail, similarly to Groucho/TLE whose tethering to enhancers depends on binding to hypoacetylated histone H3 and H4 tails. Interestingly, Pygo's histone binding requires at least one methyl group at K4-the hallmark of poised enhancers. Indeed, Drosophila Pygo is highly unorthodox due to an architectural change in its histone-binding surface that allows it to recognize asymmetrically di-methylated arginine 2-a hallmark of silent chromatin. Thus, the rare unorthodox Pygo proteins may recognize silent enhancers even earlier, long before their activation, consistent with the early embryonic function of Pygo, prior to Wg signaling (Fiedler, 2015).
Overcoming the OFF state imposed on the enhancer by Groucho/TLE involves Pygo-dependent capturing of β-catenin/Armadillo, which recruits various transcriptional co-activators to its C-terminus. Although these include CREB-binding protein (CBP), a histone acetyl transferase, its tethering to TCF enhancers is likely to co-depend on CRE-binding factors (CREB, c-Fos) and SMAD which synergize with Armadillo to activate these enhancers-similarly to the interferon-β enhanceosome where CBP recruitment also co-depends on multiple enhancer-binding proteins (Panne, 2007). The ensuing acetylation of the Wnt enhancer chromatin could promote the eviction of Groucho/TLE whose chromatin anchoring is blocked by acetylation of histone H3 and H4 tails, thus initiating the ON state (Fiedler, 2015).
Osa antagonizes Wg responses throughout development, and represses UbxB through its CRE, which also mediates repression in response to high Wg signaling. Osa could therefore terminate enhancer activity, by displacing HAT-recruiting enhancer-binding proteins such as CREB and c-Fos from CREs and by cooperating with repressive enhancer-binding proteins such as Brinker (a Groucho-recruiting repressor that displaces SMAD from UbxB) to re-recruit Groucho/TLE to the enhancer, thereby re-establishing its OFF state. Notably, Osa binds Chip, to repress various Wg and ChiLS targets including achaete-scute and dLMO (Fiedler, 2015).
Therefore, ChiLS is not only a coincidence detector of multiple enhancer-binding proteins and NPF proteins, but also a switch module that exchanges positively- and negatively-acting enhancer-binding proteins (through LID) and NPF factors, to confer signal-induced activation, or re-repression. Its stoichiometry and modularity renders it ideally suited to both tasks. It is noted that the interferon-β enhanceosome does not contain a similar integrating module, perhaps because it is dedicated to a single signaling input (Fiedler, 2015).
ChiLS is essential for activation of master-regulatory genes in the early embryo, similarly to DNA-binding pioneer factors such as Zelda (in the Drosophila embryo) or FoxA (in the mammalian endoderm) which render enhancers accessible to enhancer-binding proteins. Moreover, ChiLS maintains HOX gene expression throughout development, enabling Wg to sustain HOX autoregulation, a mechanism commonly observed to ensure coordinate expression of HOX genes in groups of cells (Fiedler, 2015).
Another hallmark of pioneer factors is that they initiate communication with the basal transcription machinery associated with the promoter. Chip is thought to facilitate enhancer-promoter communication, possibly by bridging enhancers and promoters through self-association. Indeed, Ldb1 occupies both remote enhancers and transcription start sites (e.g., of globin genes and c-Myb), likely looping enhancers to the basal transcription machinery at promoters which requires self-association, but possibly also other factors (such as cohesin, or mediator) (Fiedler, 2015).
It is noted that the chromatin association of Ldb1 has typically been studied in erythroid progenitors or differentiated erythroid cells, following activation of erythoid-specific genes. It would be interesting (if technically challenging) to examine primitive cells, to determine whether ChiLS is associated exclusively with poised enhancers prior to cell specification or signal responses (Fiedler, 2015).
Previous genetic analysis in Drosophila has linked chip predominantly to Notch-regulated processes. Likewise, groucho was initially thought to be dedicated to repression downstream of Notch, before its role in antagonizing TCF and Wnt responses emerged. Moreover, Lozenge facilitates Notch responses in the developing eye, and in hematocytes. Indeed, the first links between Groucho/TLE, RUNX and nuclear Wnt components came from physical interactions, as in the case of ChiLS. The current work indicates that these nuclear Notch signaling components constitute the Wnt enhanceosome. Although the most compelling evidence for this notion is based on physical interactions, the genetic evidence from Drosophila is consistent with a role of ChiLS in Wg responses (Bronstein, 2010). Indeed, mouse Ldb1 has been implicated in Wnt-related processes, based on phenotypic analysis of Ldb1 knock-out embryos and tissues. Notably, Ldb1 has wide-spread roles in various murine stem cell compartments that are controlled by Wnt signaling (Fiedler, 2015).
An interesting corollary is that the Wnt enhanceosome may be switchable to Notch-responsive, by NPF factor exchange and/or LMO-mediated enhancer-binding protein exchange at ChiLS. Hairy/Enhancer-of-split (HES) repressors could be pivotal for this switch: these bHLH factors are universally induced by Notch signaling, and they bind to ChiLS enhancers to re-recruit Groucho/TLE via their WRPW motifs. HES repressors may thus be capable of re-establishing the OFF state on Wnt enhancers in response to Notch (Fiedler, 2015).
Notably, restoring a high histone-binding affinity in Drosophila Pygo by reversing the architectural change in its histone-binding surface towards human renders it hyperactive towards both Wg and Notch targets even though pygo is not normally required for Notch responses in flies. Humanized Pygo may thus resist the Notch-mediated shut-down of the Wnt enhanceosome, owing to its elevated histone affinity that boosts its enhancer tethering, which could delay its eviction from the enhanceosome by repressive NPF factors. The apparent Notch-responsiveness of the Wnt enhanceosome supports the notion that orthodox Pygo proteins (as found in most animals and humans) confer both Wnt and Notch responses (Fiedler, 2015).
Previous genetic studies have shown that the components of the Wnt enhanceosome (e.g., TCF, RUNX, ChiLS and LHX) have pivotal roles in stem cell compartments, as already mentioned, suggesting a universal function of this enhanceosome in stem cells. It is therefore hardly surprising that its dysregulation, that is, by hyperactive β-catenin, is a root cause of cancer, most notably colorectal cancer but also other epithelial cancers. Indeed, genetic evidence implicates almost every one of its components (as inferred from the fly counterparts) in cancer: AML1 and RUNX3 are tumour suppressors whose inactivation is prevalent in myeloid and lymphocytic leukemias, and in a wide range of solid tumors including colorectal cancer, respectively. Likewise, ARID1A is a wide-spread tumor suppressor frequently inactivated in various epithelial cancers. Furthermore, many T-cell acute leukemias can be attributed to inappropriate expression of LMO2. Intriguingly, AML1 and ARID1A behave as haplo-insufficient tumor suppressors, consistent with the notion that these factors compete with activating NPF factors such as Pygo2, RUNX2 and possibly MACC1 (predictive of metastatic colorectal cancer) for binding to ChiLS, which will be interesting to test in future. The case is compelling that the functional integrity of the Wnt enhanceosome is crucial for the avoidance of cancer (Fiedler, 2015).
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
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