runt
Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding
factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to
increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the
stimulating effect of mammalian PEBP2/CBF beta indicates that flies are likely to have a
homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting
proteins, two Drosophila genes have been identifed: Brother and Big-brother. These genes have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirm the functional homology of Brother, Big-brother, and PEBP2/CBF beta proteins, and demonstrates that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins, both in the presence and absence of their partners, has been examined. Runt domain proteins bend DNA: this bending is influenced by Brother protein family members,
supporting the idea that heterodimerization is associated with a conformational change in the Runt
domain. Analysis of expression patterns in Drosophila embryos reveals that Brother and Big-brother
are likely to interact with Runt in vivo and further suggests that the activity of these proteins is not restricted to their interaction with Runt (Grolling, 1996).
The Drosophila gene runt plays multiple roles during embryogenesis, including one
as a pair-rule class segmentation gene. The Runt protein contains an evolutionarily conserved
domain (the Runt domain) that is found in several mammalian proteins including the human protein
AML1, which is involved in many chromosome translocations associated with leukaemia. Specific
DNA binding activity of a mammalian Runt domain is enhanced by a partner protein called
PEBP2beta/CBFbeta. DNA binding activity of Drosophila Runt is also stimulated by this protein,
suggesting the existence of a similar Runt partner protein in Drosophila. Two closely linked Drosophila genes have been cloned: runt domain partner (rp) beta1 and beta2. They
encode homologs of mouse PEBP2beta/CBFbeta. They are highly homologous to each other and to
their mammalian counterpart. Either of the rpb proteins is capable of forming a complex with Runt and
stimulating its DNA binding activity, but their temporal and spatial distributions are quite dissimilar,
suggesting that functional specificity of Runt may be conferred by the interacting partner. Runt
represses transcription dominantly when coexpressed with either partner in cultured cells, a function
consistent with a direct role for Runt in regulating expression of the even-skipped gene in Drosophila
embryos. It is concluded that Drosophila Runt can interact with either of two Runt domain partners, and
the resulting complex functions as an active repressor of transcription (Fujioka, 1996).
Runt domain family members are defined based on the presence of the 128-amino-acid
Runt domain, which is necessary and sufficient for sequence-specific DNA binding. There exists an evolutionarily
conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. However, the interaction
is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all
Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of
Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of
one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by
Runt domain proteins (Aronson, 1997).
Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are
known to activate transcription. The distinction between the Runt domain consensus (VWRPY) and the Hairy-related/HES consensus (WRPW) raises a question: are the C-termini of these families interchangeable? The ability of Runt domain proteins to activate transcription suggests that the interaction with Groucho is regulated: when Runt domain proteins assemble on a promoter that is to be activated, Groucho must either be absent or in a context where it cannot exert its repressive effects. The difference between the Groucho-recruiting C-termini of the Hairy-related/HES family and the Runt domain family may be the difference between a constitutive Groucho interaction and one that is regulated (Aronson, 1997).
Brother and Big brother were isolated as Runt-interacting
proteins and are homologous to CBFb, which interacts with
the mammalian CBFa Runt-domain proteins. In vitro
experiments indicate that Brother family proteins regulate
the DNA binding activity of Runt-domain proteins without
contacting DNA. In both mouse and human there is genetic
evidence that the CBFa and CBFb proteins function
together in hematopoiesis and leukemogenesis. Functional interactions between Brother
proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt
interaction with Brother proteins but does not affect DNA
binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays.
The
most sensitive targets of Runt are
the odd-numbered stripes of
engrailed (en) expression. Ectopic expression of the altered
Runt[G163R] has no discernible
effect on en expression,
even at levels that are fivefold
greater than required for
repression of en by the wild-type
Runt protein. To determine
whether Runt[G163R] retains any
residual activity a heat-shock
driven ectopic expression
assay was used. The high levels of Runt
expression obtained by this
method cause alterations in the
expression of other pair-rule
genes in addition to en. Even under
these conditions, the pattern of en expression as well as that of
even-skipped and fushi tarazu in embryos expressing
Runt[G163R] is indistinguishable from that of wild-type
embryos. These results indicate
that Runt[G163R] is incapable of regulating expression of
several of Runts targets in the pathway of segmentation (Li, 1999).
Interestingly, this mutant protein acts dominantly to
interfere with the Runt-dependent activation of Sex-lethal
transcription. To investigate further the requirements for
Brother proteins in Drosophila development, an
examination was carried out of the effects of expression of a Brother fusion protein
homologous to the dominant negative CBFb::SMMHC
fusion protein that is associated with leukemia in humans.
This Bro::SMMHC fusion protein interferes with the
activity of Runt and a second Runt domain protein,
Lozenge. The effects of lozenge
mutations on eye development are suppressed by
expression of wild-type Brother proteins, suggesting that
Brother/Big brother dosage is limiting in this
developmental context. Results obtained when Runt is
expressed in developing eye discs further support this
hypothesis. These results firmly establish the importance of
the Brother and Big brother proteins for the biological
activities of Runt and Lozenge, and further suggest that
Brother protein function is not restricted to enhancing
DNA-binding (Li, 1999).
Repression of Sex lethalPe expression was observed in female embryos
injected with Runt[G163R] mRNA. This dominant negative
activity indicates that the Runt[G163R] protein interacts with
some other factor(s) in the Drosophila embryos in a manner
that interferes with the activity of the wild-type Runt protein.
In contrast to this, no dominant negative interference is
observed when runt[CK], a Runt derivative that is specifically
impaired for DNA-binding, is used in this assay. If the Runt[G163R] protein is interfering by
competing for interaction with some other limiting protein
factors then Runt[CK] protein would also be expected to
behave as a dominant negative. Taken together, these results
suggest that DNA binding is required for the dominant negative
activity of Runt[G163R]. This is somewhat surprising as the
prevailing view, primarily from in vitro experiments, has been
that the central function of the Bro/Bgb and CBFbeta proteins is
to enhance DNA-binding by the Runt domain proteins. The data in this paper strongly suggest that the Bro proteins have other functions
in addition to enhancement of DNA binding by Runt. What
then might be the other functions of the Bro/Bgb proteins? One
possibility is that Bro induces a conformational change in Runt
that is required for transcriptional activation. Runt/Bro
complexes induce a bend in DNA that is greater than that
observed by the binding of Runt alone.
Perhaps DNA-bending is critical for interactions between Runt
and other transcription factors on the Sex lethalPe promoter. An
alternative possibility is that Bro/Bgb may be a bridge between
Runt and other proteins that are critical for transcription
regulation. In this model Runt[G163R] would compete for
binding to the early promoter region of Sex lethalPe-lacZ but when
bound would fail to activate transcription because other Bro-interacting
proteins are not recruited. In a two-hybrid screen
for Bro-interacting proteins a number of
proteins have been identified that appear to be members of the Trithorax group
of transcriptional regulators
(G. Golling, personal
communication to Li, 1999) have been identified. Trithorax group proteins have been
implicated as having widespread roles in transcription
activation in Drosophila development and it is attractive to
speculate that recruitment of such proteins by Runt and Bro
contributes to the activation of Sxl transcription. It is clear from
the results presented here that interactions between Runt
domain proteins and Bro/Bgb/CBFbeta proteins are important for
the functions of these conserved transcriptional regulators.
Experiments that further address the functions of the Bro/Bgb
and CBFbeta proteins will be essential for understanding the
mechanisms that account for the pivotal regulatory roles of
these proteins in diverse developmental contexts (Li, 1999).
The Core Binding Factor is a heterodimeric transcription factor complex in vertebrates that is composed of a DNA binding alpha-subunit and a non-DNA binding ß-subunit. The alpha-subunit is encoded by members of the Runt Domain family of proteins and the ß-subunit is encoded by the CBFß gene. In Drosophila, two genes encoding alpha-subunits, runt and lozenge, and two genes encoding ß-subunits, Big brother and Brother, have been identified. A sensitized genetic screen was used to isolate mutant alleles of the Big brother gene. Expression studies show that Big brother is a nuclear protein that co-localizes with both Lozenge and Runt in the eye imaginal disc. The nuclear localization and stability of Big brother protein is mediated through the formation of heterodimeric complexes between Big brother and either Lozenge or Runt. Big brother functions with Lozenge during cell fate specification in the eye, and is also required for the development of the embryonic PNS. ds-RNA-mediated genetic interference experiments show that Brother and Big brother are redundant and function together with Runt during segmentation of the embryo. These studies highlight a mechanism for transcriptional control by a Runt Domain protein and a redundant pair of partners in the specification of cell fate during development (Kaminker, 2001).
Sensitized genetic screens have proved to be powerful tools in identifying interacting proteins that participate in many different developmental pathways. A particularly impressive use of this technique in the Drosophila eye has led to the identification of the mutations in the components of the RTK pathway. Such a screening technique was used to generate mutations in genes that function with lz during eye development. The identification of mutations in a direct transcriptional target of Lz, D-Pax2, and the gene encoding a binding partner of Lz, Bgb, suggests that this screen is able to detect proteins whose function is directly related to that of Lz (Kaminker, 2001).
In this screen, two alleles of hsp83 were isolated as dominant enhancers of lzts1. Drosophila Hsp83 is a chaperone protein that has been shown to physically interact with Raf. Mutations in hsp83 were identified as downstream modifiers of the sevenless and EGFR RTK pathways. Recent studies have indicated an extensive collaboration between RTK pathways and Lz in the regulation of direct target genes such as D-Pax2 and pros. It is therefore likely that hsp83 strengthens the RTK signal transduction cascade that functions with Lz in the regulation of target genes. In addition, HSP90, the mammalian homolog of hsp83, has been shown to associate with a variety of different transcription factors and has also been proposed to function in nuclear transport. An analysis of the relationship between Hsp83 and Lz/Bgb might provide insight into the mechanism by which this transcription factor complex is translocated to the nucleus (Kaminker, 2001).
The screen also uncovered two alleles of osa/eld, a member of the brahma (brm) complex, involved in chromatin remodeling. The identification of osa as a dominant enhancer suggests that Lz may have a function related to chromatin remodeling. This is not surprising since other Runx family members are thought to function in this manner. For example, Runx2 binding has been implicated in the remodeling of the rat osteocalcin promoter. Additionally, during myeloid differentiation, Runx1 has been shown to interact with p300/CBP, a protein involved in histone acetylation. Further, Drosophila Run has been shown to bend DNA and is likely involved in modifying the architecture of target enhancers. In the eye, Lz is essential for pre-patterning an undifferentiated population of cells and preparing them to activate different target genes in response to signal transduction cascades. It is possible that this process involves remodeling of the individual enhancers through the mediation of an Osa/Lz complex. The identification of osa as a genetic modifier of lz suggests the need for future biochemical experiments to establish if such protein complexes are indeed formed during development (Kaminker, 2001).
This paper focused on the function of the partner proteins since mutations in Bgb were identified as modifiers of lz. The similarity in the phenotype of lzts1; BgbD/Df(3L)BgbK4 mutants to the null allele of lz suggests an absolute functional requirement of the partner protein during eye development. Similarly, ds-RNA interference results suggest that both partner proteins are able to function with Run during embryonic pattern formation (Kaminker, 2001).
It remains to be proven whether the disorganization seen in the PNS of Bgb can be attributed to Bgb function with the known Runt domain proteins. Similar PNS defects are seen in run mutants, but these phenotypes are difficult to interpret because of the additional segmentation phenotypes that could indirectly affect PNS development. It remains possible that Bgb functions with an as yet uncharacterized RD protein in the PNS. Consistent with this explanation, a survey of the sequence of the Drosophila genome reveals two additional runt domain proteins (Kaminker, 2001).
S2 cell expression data show that Bgb is translocated to the nucleus only in the presence of Lz. Although Bgb has a nuclear localization signal (NLS), these data suggest an additional requirement of Lz binding for its transport to the nucleus. Similar regulation of nuclear transport has been reported with Single-minded (Sim) and Tango (Tgo) heterodimers as well as with Homothorax (Htx) and Extradenticle (Exd) heterodimers. In these examples, the localization to the nucleus of either Tgo or Exd, depends on the presence of Sim or Hth, respectively. Recent work has shown that Hth binding allows nuclear transport of Exd by simultaneously inhibiting its nuclear export signal (NES) while activating its NLS. Bgb does not have a leucine-rich sequence typically associated with an NES; co-localization into the nucleus in this case is likely to involve an unmasking of the NLS causing its exposure to the transport machinery. Obviously, nuclear localization of both the alpha- and the ß-subunit is a prerequisite for activation of transcription. In fact, in human AML caused by Inv(16), the CBFß fusion protein is exclusively retained within the cytoplasm (Kaminker, 2001).
The Lz/Bgb complex provides an interesting example of post-translational stabilization of proteins through the formation of heterodimeric complexes. While the possibility that low levels of Bgb protein remain in the cytoplasm of the cell in a lz mutant background cannot be ruled out, the likely explanation for the Bgb protein not being detectable in the absence of Lz or Run is that the ß-subunit is degraded in the absence of the alpha-partner. Similar mechanisms involving degradation of a subunit operate in creating stable Exd/Hth and Sim/Tgo complexes. Tissue lacking Hth or Sim will cause degradation of Exd and Tgo, respectively. As an interesting contrast to these results, in mammalian systems it is the alpha-subunit, Runx1, that is stabilized by CBFß. In this case, the absence of the ß-partner causes a proteosome-mediated degradation of the alpha-subunit (Kaminker, 2001).
The initial cloning of Bro and Bgb raised the possibility that these genes might function redundantly during development. Although there is a stretch of 156 amino acids at the N terminus of Bgb that is not present in Bro, these proteins are 59% identical throughout the remainder of their sequence. Furthermore, Bro and Bgb have overlapping expression domains during embryogenesis. ds-RNA-mediated genetic interference experiments used in this study clearly show that Bro and Bgb function redundantly during development as heterodimeric partners of Run. A loss-of-function phenotype equivalent to a complete run null allele is revealed only in the absence of both Bro and Bgb (Kaminker, 2001).
The two partner proteins do not function redundantly in all tissues. This is highlighted by the fact that Bgb mutants have a PNS defect on their own. Thus, at least in this tissue, Bgb function is not redundant with that of Bro. This is different from redundant gene pairs such as BarH1 and BarH2 which are co-regulated in all tissues and always function together. It is also interesting to note that injection of ds-Bro generates a fairly strong segmentation phenotype, while injection of ds-Bgb does not affect segmentation patterning at all. Therefore, it is possible that in the wild-type fly, when both partners are present, Run preferentially functions with Bro. However, only in the absence of Bro, can compensation of Run function be achieved through its binding Bgb. A comparable situation exists in mice. The paralogs Hoxa3 and Hoxd3 are expressed in the same tissue, but clearly have distinct functional requirements. Yet, a compensating mechanism can be created in a background when one of the two genes is eliminated (Kaminker, 2001).
Detection of mutations in genes that function redundantly poses a difficult challenge to genetic analysis. The data show that at least for the case in study, dosage-sensitive screens involving sensitized genetic backgrounds can be used for the purpose of identifying redundant genes. Bro and Bgb together can be considered to contribute 4 copies of the partner gene. Loss of 1 out of these 4 copies in a sensitized background (lzts1; Bgb- Bro+/Bgb+ Bro+) gives rise to a detectable eye phenotype. Yet, loss of 2 copies in a wild-type background (lz+; Bgb- Bro+/Bgb- Bro+) does not generate a mutant phenotype. This remarkable sensitivity to dosage suggests that properly sensitized genetic screens could be used in the detection of redundant gene function (Kaminker, 2001).
Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. A new RNA anchoring assay in living Drosophila blastoderm embryos has been used to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. This study proposes a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule (Delanoue, 2005).
This study has used a specific RNA anchoring assay to distinguish between the four main models that could explain how apical wg and pair-rule mRNA (runt, and fushi tarazu) are retained in the apical cytoplasm after their transport by dynein. The models that have been proposed could also apply to other molecular motors and their various cargos. (1) The dynein motor could release the RNA cargo at its final destination, allowing the RNA to bind to an actin-dependent static anchor and the motor to participate in further transport. (2) The anchor could be MT associated rather than actin based. (3) RNA could be retained in the apical cytoplasm by continuous active transport without anchoring. (4) The motor itself could retain the cargo and turn into a static anchor when it reaches the final destination (Delanoue, 2005).
At the outset of this study, it was anticipated that cargo anchoring via actin was the most likely possibility given that actin is thought to be involved in anchoring of many other RNAs. It was also thought that after a motor completes a transport cycle, it releases the cargo and is available for transport of new cargo. However, in general, there has not been very good direct evidence showing that such a model is correct because of the lack of an assay that could discriminate between the transport and anchoring steps. In this study, two specific assays were used: one for transport and another for anchoring. Both anchoring and transport were assayed at the same time in the same embryo using two distinct RNAs. These specific assays have allowed a test and refutation of the prevailing actin anchoring model at least in the case of runt, fushi tarazu and wg apical mRNA localization in the Drosophila blastoderm embryo. Against expectations, the results show that the fourth model is correct, namely that wg and pair-rule RNA are anchored by a dynein-dependent mechanism so that the motor molecules are maintained to the site of anchoring with the cargo. The data shows that the requirement for dynein to anchor the apical RNA is independent of the ATPase activity of the motor and its transport cofactors Egl and BicD, all of which are required for the active transport of the RNA. These observations are best explained by a model in which the dynein motor involved in apical transport of RNA does not release the cargo and acts as a static anchor at the final destination (Delanoue, 2005).
It is interesting to consider how a dynamic motor such as dynein could turn into a static anchor after completion of cargo transport. Dynein is a large multicomplex motor that is difficult to work with in vitro. Nevertheless, many of the subunits of dynein are defined and the force-generating protein, Dhc, is thought to contain physically distinct ATPase and MT binding domains. It is therefore easy to imagine how the motor could change to a static anchor by remaining attached to MTs via the MT binding domain and losing its ATPase force-generating capacity. Indeed, ATPase-independent MT binding has been observed with dynein under in vitro conditions. While it is difficult to compare in vitro studies with the current studies in vivo, the latter are likely to show much more complex and varied interactions with proteins in the cell. Indeed, anchoring may also involve interactions with additional components not present in vitro, such as MT-associated proteins (MAPs), which could stabilize the binding of dynein to the apical MTs or could physically obstruct the motor movement. Another possibility could be anchoring through association with ribosomes, but this can be ruled out in the case of wg and pair-rule RNA, since RNAs lacking a coding region can be transported and anchored correctly. Alternative hypotheses, which cannot be ruled out, include a change of conformation or modifications of the structure of the dynein-dynactin complex. While the data demonstrate conclusively a new RNA-anchoring function for dynein, they do not allow distinguishing between the various hypotheses of how this anchoring occurs at the molecular level, nor test definitively whether Dynactin is required for anchoring. p50/dynamitin is present with the anchored RNA, and overexpression of p50/dynamitin and a Glued/p150 allele cause a partial inhibition of RNA localization with no obvious effects on anchoring. These results suggest, but do not demonstrate conclusively, that Dynactin is not required for anchoring. Furthermore, while it is shown that the ATPase activity of the motor is not required for anchoring, this observation does not test whether dynactin is required in addition to dynein for anchoring (Delanoue, 2005).
Whatever the molecular basis for the dynein anchoring function that was uncovered, it seems likely that the described anchoring does not involve a single dynein molecule anchoring a single RNA molecule. Instead, the RNA cargo is likely to consist of particles containing many RNA molecules and probably many motor complexes. The cargo is thus likely to remain strongly attached to at least some motor molecules throughout transport and anchoring. However, it is not yet known what the linkers between the RNA and motors are (Delanoue, 2005).
Little is also known about the mechanism of anchoring of other dynein cargos, although the mechanism of transport of RNA by dynein could be very similar to other cargos such as lipid droplets. Dynein is also required for nuclear positioning and tethering in many systems, so its role as a static anchor may be widespread. Furthermore, some kinesin-like proteins are also thought to interact with static cell components, and recent in vitro studies show that myosin VI can switch from a motor to an anchor under tension. This process has been proposed to stabilize actin cytoskeletal structures and link protein complexes to actin structures. It is therefore proposed that myosins, kinesins, and dynein may all be able to switch under certain circumstances from dynamic motors to static anchors and that the observations of this study may represent a general principle for anchoring of some cargos following transport to their final cytoplasmic destination (Delanoue, 2005).
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).
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runt :
Biological Overview
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