Gene Families

The Interactive Fly

Zygotically transcribed genes

Antennapedia and Bithorax Complexes

Intergenic transcription through a bithorax-complex polycomb group response element counteracts silencing

Interactions among Polycomb domains are guided by chromosome architecture

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression

Polarized regulatory landscape and Wnt responsiveness underlie Hox activation in embryos

Genes of the Antennapedia complex

Genes of the bithorax complex
Cofactors for Hox complex genes

'Homeobox' antennapedia-type protein signature

The six Drosophila proteins that belong to the antennapedia-type Homeobox subfamily are Antennapedia (ANTP), Abdominal-A (ABD-A), Deformed (DFD), Proboscipedia (PB), Sex combs reduced (SCR) and Ultrabithorax (UBX). The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the'Homeobox' antennapedia-type protein signature.

Intergenic transcription through a bithorax-complex polycomb group response element counteracts silencing

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, it was shown that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system (Schmitt, 2005; full text of article).

Intergenic transcription in the BX-C has a profound phenotypic effect on the expression of the Hox genes. The results with transgenes suggest that the spatially and temporally regulated transcription of noncoding RNAs in the BX-C induces a remodeling of the chromatin, thereby preventing PcG-mediated silencing. The consequence of this is the segment-specific activation of the Hox genes. This is probably especially important in large gene complexes where PREs are located at long distances from the promoters they regulate. It has been shown that the activation of a minimal 219-bp Fab-7 PRE is not accompanied by transcription through the element. However, in this case the minimal PRE was juxtaposed to the promoter, probably benefiting from the open chromatin environment induced by the bound transcription factors. Other results indicate that an 870-bp large Fab-7 PRE, under similar conditions but containing more PcG protein-binding sites, cannot be activated anymore. This suggests that over a certain threshold level of silencing, imposed by the stability of the silencing complexes, chromatin remodeling by transcription is required to remove PcG complexes in order to counteract their silencing activity in a mitotically heritable fashion (Schmitt, 2005).

Hox cluster regulation is only part of the entire PcG/trxG memory system, prompting an analysis of the transcription pattern of characterized en and predicted salm, slou, and tll target genes as well as their respective PRE sequences. RNA in situ hybridizations reveal that these sequences are also transcribed in a pattern that reflects the expression of the cognate target genes. This suggests that the mechanism of epigenetic activation of PREs initially described for the homeotic gene clusters may be required for the regulation of many more PcG target genes than previously thought. Transcription through these PREs can be either uni- (tll PRE) or bidirectional (en, slou, and salm PREs), further suggesting that the induction of epigenetic activation relies on the remodeling of chromatin induced by the transcriptional process, rather than by the noncoding RNAs (Schmitt, 2005).

With the transgenic reporter system, it was shown that the constitutive transcription through the Fab-7 PRE from the actin5c promoter results in the stable epigenetic activation of this element. This raises the question of how this could be achieved mechanistically. As has been proposed for the developmental regulation of globin gene expression and the regulation of VDJH-recombination in mice, the opening of the chromatin structure at a transcribed PRE may be induced by the passing of the transcriptional machinery through the regulatory sequence. It has been shown that the elongating RNA polymerase II complex is associated with the SWI/SNF remodeling complex and a histone acetyl transferase activity. Such enzymatic activities linked with the transcription machinery may catalyze the epigenetic modification of chromatin (Schmitt, 2005).

In this respect, it is interesting to note that most of the trxG mutants were initially uncovered as suppressors of the Pc phenotype, and that the combination of PcG with trxG mutations can restore the typical phenotype of the single mutations to wild type. The molecular mechanism behind this antagonism is not clear. Using clonal analysis, it has been shown that the trxG proteins Ash1 and Trx do not function as coactivators of Hox gene expression, but that they are required as anti-repressors to prevent PcG-induced silencing. In contrast, the Brahma complex containing the trxG proteins Brm, Osa, and Mor to acts as a coactivator of transcription, and a subset of trxG genes encode components of the Mediator complex. The finding that transcription through Fab-7 induces the epigenetic activation of this PRE may explain how trxG complexes involved in more general transcriptional processes antagonize the establishment of PcG silencing (Schmitt, 2005).

Interestingly, in budding yeast, intergenic transcription through a promoter has been shown to prevent the binding of a transcriptional activator to its target sequences. It is possible that, in a similar fashion, the transcription through PREs may lead to the displacement of repressive PcG complexes from the chromatin and/or the prevention of PcG recruitment to the PRE in the first place. Enzymatic activities carried by the RNA polymerase II complex may subsequently or in addition modify histones at PREs with positive epigenetic marks like acetyl or methyl moieties. Interestingly, has been shown that the sequential induction of HoxB gene expression in mouse embryonic stem (ES) cells by retinoic acid correlates with the orchestrated looping out of this locus from chromosome territories. This indicates that, in addition to locus-wide changes in the chromatin structure such as histone modifications, the transcriptional activity of genes may be regulated by an additional order of complexity. It is possible that, in addition to inducing 'small-scale' changes in the chromatin structure, transcription through PREs may lead to a subnuclear relocation of the target gene locus from a repressive into a transcriptionally permissive environment (Schmitt, 2005).

Removal or inhibition of binding of PcG silencing complexes to PREs by tissue-specific transcription appears to be an attractive mechanism to counteract the constant pressure of the repressive system acting by default. However, with such a solution, the problem of epigenetic maintenance is simply moved to another level, since the question arises of what prevents the intergenic transcription from being silenced by the PcG. The simple answer -- promoters of noncoding transcripts are not sensitive to PcG/PRE silencing -- is probably not valid. Noncoding transcripts of the BX-C are activated by the same set of early segmentation genes as the Hox protein encoding mRNAs. As such, their subsequent regulation might be subjected to the same regimen of factors as the protein-encoding transcripts. However, the problem of transcriptional memory might be reduced to the problem of how to inhibit PcG silencing in particular cells/tissues, while the rest will be down-regulated by default (Schmitt, 2005).

With the processive transcription as the central issue, the cell cycle might become an important factor for the maintenance of active transcription. In Drosophila, PcG proteins dissociate from the chromosomes at mitosis. Thus, if after mitosis intergenic transcription starts before PcG proteins rebind to the PREs, the chromatin would be turned into an active mode, and would thus be protected from PcG-mediated silencing until the next round of cell division. At this point, it remains an open question whether continuous transcription of noncoding RNAs is required throughout the cell cycle to prevent the PcG complexes from rebinding, or whether the initial setting of positive epigenetic marks by the early transcription process is sufficient to prevent silencing during interphase. This proposed mechanism further reduces the problem of how transcriptional activity is maintained to the problem of how only a positive epigenetic mark is maintained during DNA replication and mitosis. Here, recent advances in studies of histone variants propose some attractive candidate marks. In particular, the histone variant H3.3 associated with the transcription of active genes could be envisaged as a possible positive signal that is locally maintained and propagated at cell division. As has been suggested before, targeted deposition of the H3.3 variant at sites of active transcription may serve to remove repressive epigenetic marks such as methylation. The establishment of stable PcG silencing complexes not only requires a sequence component, but is also accompanied by the methylation of K9 and K27 of histone H3. In contrast, positive marks, which have been shown to be mainly associated with H3.3 compared to H3, would be specifically enriched at a transcribed PRE and transmitted through mitosis. After cell division, these epigenetic marks may then in turn provide a platform for noncoding transcription through the PRE early in the cell cycle, which itself may re-establish the full active chromatin environment, unsuitable for PcG protein binding and silencing. Additionally, the reported result that an activated PRE is still maintained over a certain period after transcription has ceased (by removal of the promoter by the inducible Cre recombinase) suggests that this positive epigenetic signal is quite stable and is only diluted out by multiple cell divisions (Schmitt, 2005).

In summary, it is proposed that transcriptional maintenance during development by the PcG/trxG system is primarily a process of preventing PcG silencing to occur at those target genes that need to be maintained active in a defined cell lineage. The advantage of this mode of action is that a positive epigenetic mark, surviving DNA replication and mitosis, is sufficient to ensure stable and heritable maintenance of gene expression patterns, since the silenced mode is created by default. As such, transcription of intergenic sequences would serve as an anti-silencing mechanism that would continually counteract the initiation of this PcG-mediated silencing. In the future, it will be important to pursue the involvement of the various trxG components in the establishment and maintenance of regulatory transcription mechanisms and to analyze the link to cell cycle control and the identity and propagation of the positive epigenetic marks required to sustain active transcription (Schmitt, 2005).

Interactions among Polycomb domains are guided by chromosome architecture

Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. This study adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. The results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and it was demonstrated that a topological rather than a sequence-based mechanism is responsible for this constraint. These results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture (Tolhuis, 2011).

This study successfully adapted the 4C method to systematically map long-range chromatin contacts with limited material from a single fly tissue. With this method, interactions were detect between the ANT-C and BX-C in central brain. This observation is in good agreement with earlier microscopic reports, underscoring the strength of the method. Importantly, it was further demonstrated that not only the two Homeotic gene clusters, but also many other PcG target genes interact, suggesting that long-range chromatin contacts between PcDs are common in central brain tissue. The control fragments (wntD, CG5107, Crc, and RpII140), which do not reside in PcDs, have interactions that are distinct from PcDs, emphasizing the specificity of the findings (Tolhuis, 2011).

PcG targets show a strong preference for interaction with other PcG targets, suggesting that PcG proteins help to establish these interactions. This is in line with earlier in vivo and in vitro results that indicated that PcG proteins can keep certain DNA sequences together. However, interactions among PcG target genes are constrained by overall chromosome architecture, because the data demonstrate that loci need to be on the same chromosome arm for efficient interaction (Tolhuis, 2011).

Discrete interaction domains (DIDs) range in size from 6 to 600 kb, with an average of ~170 kb. Thus, highly local strong enrichments are found as well as moderate enrichments over larger regions, which may reflect different types of long-range interactions. It is emphasized that interactions as detected by 4C technically represent events of molecular proximity of DNA sequences, and not necessarily physical binding. Based on the current data it is therefore not possible to identify within the DIDs sequence elements that may mediate direct contact with other DIDs. It is conceivable that contacts between PcDs may occur at any position within the PcDs; if PcG protein complexes have an intrinsic propensity to aggregate, as has been observed in vitro, then large PcDs may have a higher chance of interacting with each other due to their larger ‘sticky’ surface area (Tolhuis, 2011).

The 4C method is a cell population based assay that only detects the most frequent interactions in the population. Previous 4C studies in mammalian cells suggested an extensive network of long-range interactions. The current data also suggests an extensive network among interacting PcDs. However, microscopic studies in mammalian cells revealed that specific long-range interactions occur only in a small proportion of the cells. Likewise, only a proportion of D. melanogaster cells show contacts between the two Homeotic clusters. Therefore, 4C data have to be carefully interpreted, because the identified interactions are in part stochastic and do not all occur simultaneously. As a consequence, it is not known how many PcDs interact in a single cell, but the most common interactions are known in the population of larval brain cells (Tolhuis, 2011).

Interphase chromosomes in most eukaryotes occupy distinct 'territories' inside the nucleus, with only a limited degree of intermingling. Although some studies have reported interactions between some loci that are on two different chromosomes (interchromosomal), unbiased 4C mapping in mouse tissues has indicated that interactions within the same chromosome (intrachromosomal) occur much more frequently than between different chromosomes. In addition, a recent genome-wide map of chromatin interactions in human cells showed that intrachromosomal interactions occur with higher frequency than interchromosomal contacts. The current data are in agreement with these observations, and show that most interactions are even limited to single chromosome arms, at least in D. melanogaster larval brain. Since the experiments indicate that a topological mechanism prevents interactions between the two arms of a chromosome, it is proposed that each arm (rather than the chromosome as a whole) forms a distinct territory. This is consistent with early microscopy studies of chromosome architecture in D. melanogaster, which suggested that chromosome arms are units of spatial organization (Tolhuis, 2011).

What topological mechanism may limit contacts between the two chromosome arms? About ~16 Mbp of pericentric heterochromatin are located in between the two euchromatic arms of chromosome 3. This heterochromatin region could act as a long spacer and prevent efficient interactions between DNA fragments that are located on either chromosome arm. However, the data show that interactions within one arm can span even longer distances, such as between Ptx1 and grn (~22.7 Mbp). Another explanation may be that the pericentric regions of all chromosomes assemble into a nuclear compartment, called chromocenter. This large structure could physically obstruct interactions between chromosome arms (Tolhuis, 2011).

Previous reports have demonstrated that certain PcG-bound PREs can pair in trans (i.e. when they are located on different chromosomes). First, a Fab7 PRE-element integrated on the X chromosome (Fab-X) was often found in close spatial proximity to the endogenous Fab-7 in the BX-C (chromosome 3R), although this phenomenon appears to be tissue-specific and dependent on the transgene integration site. Second, a microscopy assay based on Lac repressor/operator recognition showed that the Mcp PRE-element is able to pair with copies of that same element inserted at remote sites in the genome either in cis (i.e. when they are located on the same chromosome) or in trans. The 4C experiments also identified cases of trans interactions between endogenous loci, although they occur with low frequency (approximately 5% occurs in trans) (Tolhuis, 2011).

Although rare, such interactions between loci on different chromosome arms are of interest, because they indicate that the topological constraints imposed by chromosome architecture can in principle be overcome. In mammalian cells, there is evidence that the relative position of a gene locus within its chromosome territory (CT) influences its ability to form either cis- or trans-interactions. Peripheral regions of mammalian CTs intermingle their chromatin, which may allow for interactions between chromosomes. Indeed, more trans contacts are identified by 4C using a bait that often resides in the CT periphery compared to a bait located in the interior of a CT. Likewise, activation of the HoxB gene cluster during differentiation coincides with relocation away from its CT interior, and the active HoxB1 gene more frequently contacts sequences on other chromosomes compared to the inactive gene. In line with this, a varying degree of trans interactions are observed among eight bait sequences, suggesting distinct capacities to contact chromatin on other chromosomes. The trh gene has the strongest capacity to contact other chromosome arms. Interestingly, trh is located within 500 Kbp of the telomere of chromosome 3L, and 5 out of 6 contacts in trans occur within 500 Kbp of other telomeres. Thus, interactions between chromosome arms may be possible if loci are favorably positioned on the edge of chromosome (arm) territories, which could be the case for telomeric sequences in larval brain cells (Tolhuis, 2011).

The experiments with strain In(3LR)sep showed dramatic changes in interactions, such as loss of contacts between the Homeotic gene clusters and gain of contacts with other PcDs. Despite these changed interactions, no convincing evidence was found for global gene expression alterations on the In(3LR)sep chromosome. This raises the question: how relevant are long-range chromatin contacts between PcG-target genes for regulation of expression?

The lack of detectable expression changes may indicate that long-range interactions have only quantitatively subtle effect on the regulation of gene expression. Nevertheless, such subtle effects on gene expression could be very important for long-term viability and species survival. In(3LR)sep animals do suffer from an overall reduced viability during several stages of development, which may indicate a generally reduced fitness, possibly due to a dimly altered regulation of gene expression (Tolhuis, 2011).

Alternatively, PcG gene regulation may not be affected in strain In(3LR)sep, because Abd-B and Antp, although they no longer interact with each other, still prefer to interact with other PcDs, suggesting that it is not relevant which PcDs interact. In such a model, the complement of all interactions contributes to PcG-mediated gene silencing in a population of cells (Tolhuis, 2011).

Finally, it is interesting to note that over ~100 million years of evolution of the Drosophila genus, exchange of genes between chromosome arms has been rare despite extensive rearrangements within each arm. Chromosome arm territories ensure that genes within a single arm are relatively close compared to genes on other arms, which may have resulted in an increased chance of rearrangements within one arm. Alternatively, the importance of long-range interactions among sets of genes, which are topologically limited to the same arm, may have contributed to the selective pressure that has led to this remarkable conservation of the gene complement of each chromosome arm (Tolhuis, 2011).

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, this study shows that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, a motif in the posterior Hox protein Abdominal-A (AbdA) was identified that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism (Noro, 2011).

fkh250 is a 37-base-pair (bp) element from the forkhead (fkh) gene, which is directly regulated by Scr; it contains a single Hox-Exd-binding site that, compared with other Hox-Exd heterodimers, is preferentially bound by Scr-Exd in vitro (Ryoo, 1999). When lacZ is placed under the control of fkh250, fkh250-lacZ is specifically expressed in PS2 in an exd- and Scr-dependent manner. Indeed, misexpression of Scr throughout the Drosophila embryo can ectopically activate fkh250-lacZ. Notably, ectopic activation of fkh250-lacZ occurs even in the abdomen, in the presence of endogenous, more posterior Hox (Noro, 2011).

In contrast to fkh250, fkh250CON (for 'consensus') is an artificial variant of fkh250 with two base pair substitutions that enable fkh250CON-lacZ to be directly regulated by four Hox genes in an exd-dependent manner: Scr, Antp, and Ubx activate this reporter in PS2-PS6, while AbdA represses it in abdominal segments (Ryoo, 1999). Consistent with its relaxed specificity in vivo, fkh250CON binds well to Scr-Exd, Antp-Exd, Ubx-Exd, and AbdA-Exd heterodimers in vitro. The promiscuous binding and regulation by multiple Hox proteins classifies fkh250CON as a shared Hox target gene, while the Scr-specific regulation of and binding to fkh250 suggests that it is a specific Hox target gene (Mann, 2009; Noro, 2011).

Because of their distinct specificities, fkh250-lacZ and fkh250CON-lacZ provide an ideal system to examine the molecular mechanism of phenotypic suppression. In accordance with the premise of posterior dominance, coexpression of Scr and AbdA throughout the fly embryo leads to repression of fkh250CON-lacZ by AbdA. Note that both fkh250 and fkh250CON-lacZ require direct binding by the Hox cofactor Exd. The primary distinction between these two readouts is that AbdA-Exd binds well to fkh250CON but not to fkh250. Accordingly, it is concluded that AbdA cannot suppress the activities of Scr if it cannot bind to the target element. Furthermore, in this system, posterior dominance cannot be mediated by miR activity or competition for factors, such as Exd, off DNA. Rather, these data support a model in which competition for cofactor-dependent DNA binding underlies phenotypic suppression for shared Hox target genes (Noro, 2011).

If AbdA is outcompeting Scr for binding to fkh250CON, AbdA would be expected to have a higher affinity for this sequence compared with Scr. To test this prediction, the affinities of AbdA-Exd and Scr-Exd heterodimers for fkh250CON were measured in vitro. AbdA-Exd heterodimers bound more than twofold more tightly to fkh250CON compared with Scr-Exd. Thus, at the same concentration, AbdA-Exd is more likely than Scr-Exd to be bound to fkh250CON, consistent with the idea that competition depends on cofactor-dependent DNA binding (Noro, 2011).

Binding to fkh250CON is Exd-dependent for both AbdA and Scr, implying that AbdA has domains that allow higher binding affinity with Exd to this target site. In general, Hox interactions with Exd are mediated by the highly conserved, four-amino-acid motif YPWM, which directly binds to a hydrophobic pocket established by the three-amino-acid loop extension (TALE) in the Exd homeodomain (Mann, 2009). For some Hox proteins, the YPWM-TALE interaction is necessary and sufficient for cooperative DNA binding with Exd and target gene regulation in vivo (Joshi, 2010). In addition to the YPWM motif, AbdA, but not Scr, has a second well-conserved tryptophan-containing motif, TDWM, which could play a role in mediating AbdA-Exd interactions. However, when a mutant form of AbdA in which both the YPWM and TDWM motifs are mutated to alanines (2WAla) was coexpressed with Scr in the phenotypic suppression assay, fkh250CON-lacZ was repressed to the same extent as by wild-type AbdA. Thus, although the YPWM and TDWM may contribute to interactions with Exd, these motifs are not necessary for AbdA to dominate over Scr (Noro, 2011).

Immediately C-terminal to its homeodomain, AbdA contains a so-called UbdA motif, a nine-amino-acid sequence also present in Ubx, which has been suggested to mediate cooperative binding with Exd to some DNA sequences (Merabet, 2007). In fact, UbdA is part of a larger 23-residue conserved region adjacent to the AbdA homeodomain, which is referred to as the UR motif (for UbdA-RRDR). To determine whether this or other regions in the C-tail of AbdA are involved in mediating phenotypic suppression, a series of C-terminal truncations were tested for their ability to compete with Scr for the repression of fkh250CON-lacZ in vivo. All AbdA variants were epitope-tagged, allowing use of transgenes that express at similar levels (Noro, 2011).

AbdA's ability to compete with Scr for fkh250CON regulation is eliminated when the entire C terminus is removed (ΔC197). Adding back only the UR motif partially restores AbdA's ability to dominate over Scr (ΔC220). Consistently, an internal deletion that removes most of the UR motif (Δ200-220) exhibits a reduced ability to repress fkh250CON-lacZ. No additional loss of repressive activity is displayed by an AbdA variant in which both the YPWM and TDWM motifs are mutated in combination with this internal deletion (2WAlaΔ200-220). Additional sequences in the C-tail of AbdA may account for the residual activity of variants lacking the UR motif (Δ200-220 and 2WAlaΔ200-220). All AbdA variants used in this study are capable of repressing the exd-independent target gene spalt in the wing imaginal disc, confirming that these mutants are still functional transcription factors. Furthermore, these mutants retain the ability to repress gene expression in vivo, arguing that AbdA's repressive activity is not sufficient to account for its ability to dominate Scr. Together, these data highlight the importance of the UR motif for phenotypic suppression (Noro, 2011).

The above data show that the UR motif is required for AbdA to compete with Scr in vivo. To test the hypothesis that UR carries out this function by facilitating cooperative DNA binding with Exd, the ability of the truncated AbdA variants to bind fkh250CON in complex with Exd was analyzed. In general, the results correlate with the in vivo phenotypic suppression assay: Those mutants that failed to suppress Scr's ability to activate fkh250CON-lacZ (ΔC197, Δ200-220, and 2WAlaΔ200-220) were severely compromised in binding fkh250CON with Exd in vitro. Together, these data strongly suggest that cooperative DNA binding with Exd is required for phenotypic suppression and that domains unique to AbdA are critical for its ability to dominate over Scr. More specifically, they argue that AbdA's UR motif is necessary for cooperative binding of AbdA and Exd to fkh250CON and that the YPWM and TDWM motifs are not sufficient to mediate this interaction on this binding site. The insufficiency of the YPWM motifs to mediate cooperative binding with Exd has been observed for other Hox proteins, suggesting that the use of paralog-specific motifs such as UR may be a general phenomenon (Noro, 2011).

To test the generality of AbdA's dependency on its UR motif for posterior dominance, the same AbdA variants were analyzed for their ability to suppress the activity of the thoracic Hox protein Antp in the patterning of the larval epidermis. When ectopically expressed, Antp transforms the head and first thoracic segment (T1) toward the identity of the second thoracic segment (T2), where Antp is normally expressed). In contrast, when AbdA is ectopically expressed, the head and thorax acquire abdominal segmental identities. Consistent with the rules of phenotypic suppression, wild-type AbdA is able to produce this transformation even in the presence of exogenous Antp. However, similar to the results with fkh250CON-lacZ, AbdA mutants that are compromised in their ability to cooperatively bind DNA with Exd (e.g., ΔC197, Δ200-220, and 2WAlaΔ200-220) fail to suppress the activity of Antp (Noro, 2011).

Taken together, these data support a model in which phenotypic suppression depends on a competition for cofactor-dependent DNA binding. It follows that this mechanism would only apply to readouts that depend on regulatory elements that are targeted by multiple Hox proteins. For example, ectopic Scr can activate fkh and other target genes required for salivary gland development in more posterior segments, illustrating that this Hox-specific function does not obey phenotypic suppression. Furthermore, it is particularly noteworthy that, compared with the anterior Hox protein Scr, AbdA has additional motifs that facilitate complex formation with Exd on DNA. These data suggest that when phenotypic suppression is observed, the more posterior Hox proteins may have a higher affinity for shared binding sites; this higher affinity is a consequence of the quantity and quality of motifs that mediate cooperative DNA binding with Exd. It is speculated that these motifs may be used differently at different target genes and binding sites. It is suggested that the YPWM motif provides a common, basal level of interaction between Hox proteins and Exd. In the context of Hox-specific regulatory elements, this motif may be sufficient to enable Hox-Exd regulation of some target genes. In contrast, in the context of shared enhancers and when multiple Hox proteins are coexpressed, additional, paralog-specific motifs present in the more posterior Hox proteins enable tighter binding of Hox-Exd dimers to DNA, leading to more posterior phenotypes. This was shown to be the case for a single shared Hox-Exd enhancer and suggest that the generality of this mechanism for phenotypic suppression will become apparent as more shared and specific targets for Hox proteins are characterized at high resolution (Noro, 2011).

Polarized regulatory landscape and Wnt responsiveness underlie Hox activation in embryos

Sequential 3'-to-5' activation of the Hox gene clusters in early embryos is a most fascinating issue in developmental biology. Neither the trigger nor the regulatory elements involved in the transcriptional initiation of the 3'-most Hox genes have been unraveled in any organism. This study demonstrates that a series of enhancers in the mouse, some of which are Wnt-dependent (see Drosophila Wg), is located within a HoxA 3' subtopologically associated domain (subTAD). This subTAD forms the structural basis for multiple layers of 3'-polarized features, including DNA accessibility and enhancer activation. Deletion of the cassette of Wnt-dependent enhancers proves its crucial role in initial transcription of HoxA at the 3' side of the cluster (Neijts, 2016).


Neijts, R., Amin, S., van Rooijen, C., Tan, S., Creyghton, M.P., de Laat, W. and Deschamps, J. (2016). Polarized regulatory landscape and Wnt responsiveness underlie Hox activation in embryos. Genes Dev 30: 1937-1942. PubMed ID: 27633012

Joshi, R., Sun, L. and Mann, R. (2010). Dissecting the functional specificities of two Hox proteins. Genes Dev 24: 1533-1545. PubMed Citation: 20634319

Mann, R. S., Lelli, K. M. and Joshi, R. (2009). Hox specificity unique roles for cofactors and collaborators. Curr. Top. Dev. Biol. 88: 63-101. PubMed Citation: 19651302

Merabet, S., et al. (2007). A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax. Proc. Natl. Acad. Sci. 104: 16946-16951. PubMed Citation: 17942685

Noro, B., Lelli, K., Sun, L. and Mann, R. S. (2011). Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression. Genes Dev. 25(22): 2327-32. PubMed Citation: 22085961

Ryoo, H. D, and Mann, R. S. (1999). The control of trunk Hox specificity and activity by Extradenticle. Genes Dev. 13: 1704-1716. PubMed Citation: 10398683

Schmitt, S., Prestel, M. and Paro, R. (2005). Intergenic transcription through a polycomb group response element counteracts silencing. Genes Dev. 19(6): 697-708. Medline abstract: 15741315

Tolhuis, B., et al. (2011). Interactions among Polycomb domains are guided by chromosome architecture. PLoS Genet. 7(3): e1001343. PubMed Citation: 21455484

Zygotically transcribed genes

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