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Title: Multifactorial Regulation of a Hox Target Gene}
Article:
Abstract:
Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior–posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper( rpr) by the Hox protein Deformed( Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements( HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.
Introduction:
Distinct morphological structures exist along the anterior-posterior( A/P) axes of animals, and the Hox genes represent the major regulators for patterning of this body axis in organisms as diverse as fruit flies, fish and humans[1],[2],[3]. Since Hox genes code for transcription factors, Hox-dependent morphogenesis is driven by the differential regulation of downstream genes[4],[5],[6]. In line with the very diverse and many-fold effects of Hox proteins on morphogenesis, Hox transcription factors are known to regulate a large number of Hox downstream genes[7],[8], including genes that themselves have broad effects on morphology, as well as genes involved in terminal differentiation[reviewed in 3]. Hox genes are expressed in broad and partially overlapping domains along the A/P axis[1],[2],[3], and their constant and simultaneous activity within hundreds of cells is required throughout development[1]. Despite being active in a very large number of cells, Hox proteins affect target gene expression in precisely defined sub-domains in the animal[for example 9],[10],[11],[12],[13],[14],[15]. In the most extreme case the regulation of a Hox target gene can be limited to a single cell[16]. In addition, some downstream genes can be activated and repressed by the same Hox protein depending on the tissue or developmental stage. Finally, this context dependency also allows a single Hox protein to affect distinct sets of target genes in the same cells during the course of development[8],[11],[17]. While more and more of these complex regulatory interactions are being described, the molecular mechanisms underlying the spatio-temporal precision of Hox target gene regulation is only poorly understood. This is in large part due to our limited knowledge of the design and function of Hox-dependent enhancers and promoters and their interaction with the regulatory environment. Only a few Hox regulated enhancers have been analyzed in some detail in Drosophila[3]. For example, the activation of wingless( wg) expression in the visceral mesoderm of Drosophila embryos had been shown to be dependent on the Hox protein Abdominal-A( Abd-A) and the Dpp/TGF-β signalling pathway, with both activities functioning on a small wg enhancer[11]. Here, two transcriptional effectors of the Dpp/TGF-β pathway, Mother against dpp( Mad) and Creb, had been shown to mediate the Dpp response on the wg enhancer, and were thus assumed to work in concert with the Hox protein Abd-A[11]. Only recently, Mad and another effector of the Dpp/TGF-β pathway, Medea( Med), have been found to collaborate with the Hox protein Ultrabithorax( Ubx) to repress transcription of the Hox target gene spalt major( sal) in the haltere by independently interacting with adjacent Mad/Med and Ubx binding sites in a small sal enhancer[18]. And finally, two transcription factors very well known for their function in the Drosophila segmentation cascade, Engrailed( En) and Sloppy paired 1( Slp1), were shown to assist the Hox proteins Ubx and Abd-A in repressing Distal-less( Dll) expression in the abdomen of Drosophila embryos by occupying their identified binding sites in a minimal Dll enhancer[10]. Another well-studied direct Hox target gene in Drosophila is the apoptosis gene rpr, which is activated by the Hox protein Dfd in the anterior part of the maxillary segment through four binding sites located in the rpr-4S3 regulatory fragment[13]. Since Dfd is active throughout the maxillary segment, whereas rpr RNA is found only locally[19], it seemed likely that additional factors contribute to region specific Dfd-dependent rpr expression. Here, we find that eight transcriptional regulators, with diverse roles in patterning or differentiation processes, co-operate with Dfd in the regulation of rpr. Within their spatially restricted expression domains, these regulators are recruited to a minimal rpr enhancer through specific cis-regulatory DNA sequences and act together with Dfd to regulate rpr expression in the appropriate spatio-temporal pattern. Thus, our data support the idea that the combinatorial activity of Hox proteins and diverse transcriptional regulators on small regulatory elements is responsible for the spatially and temporarily restricted expression of Hox target genes in vivo. In addition, our data show that even small Hox-dependent enhancers are complex and integrate diverse regulatory inputs, which result in precise spatio-temporal expression of Hox target genes.
Discussion:
We have shown that eight transcriptional regulators, Apt, Gcm, Ems, En, Slp1, Brk, Doc1 and Disco, are required in addition to the Hox protein Dfd to properly regulate the expression of the apoptosis gene reaper in a specific subset of cells of the maxillary segment. The finding that such a large number of structurally unrelated transcription factors with important and diverse functions during differentiation and cell-type specification processes assist Dfd in the regulation of the rpr4S3/3′ enhancer element was surprising. For example, Gcm is one of the major regulators of glial cell differentiation, consistent with its expression in glial precursor cells during embryogenesis[42],[43]. Two other factors, Brk and Doc1, are both known to play important roles in the Dpp/TGF-β signalling pathway: Brk, a genuine transcriptional repressor[24], acts as a negative regulator of Dpp-dependent genes[44],[45],[46], whereas Doc1, one of the three genetically redundant Dorsocross transcription factors required for amnioserosa development in Drosophila[47], is a direct target of the Dpp pathway[48]. Furthermore, Apt and Disco have previously been found in genetic screens designed to identify modifiers/interactors of Dfd[21],[22]. Mutations in these genes, which are expressed in the gnathal segments[21],[22], result in severe malformations of structures derived from these segments, similarly to the defects observed in Dfd mutants. And finally, we have identified three factors known to be critically involved in patterning the A/P axis as important regulators of rpr expression: the gap-like segmentation gene product Ems, which is important for patterning embryonic head structures[49],[50], and two segment polarity factors, En and Slp1. Taken together, our findings suggest that proper spatio-temporal Hox target gene regulation is achieved by the combined action of multiple transcriptional regulators: the Hox proteins themselves and a large number of structurally diverse transcription factors. Although the interaction with additional transcription factors has been reported before[11],[14],[51], the finding that a multitude of diverse factors is required to regulate the activity of a small HRE adds a new layer of complexity to the mechanisms of Hox target gene regulation. Our results not only demonstrate that the newly identified factors are functionally involved in rpr regulation, but also show mechanistically that they contribute to localized rpr activation by direct interactions with specific DNA sequences located in the rpr4S3/3′ enhancer element both in vitro and in vivo. Additionally, all factors very likely bind to their target sequences independently of Dfd. Thus, our findings support and significantly extend recent observations: the repression of the Hox target sal in the haltere requires the direct interaction of the Hox protein Ubx and two Dpp downstream effectors, Mad and Med, with adjacent binding sites in the sal1. 1 CRE[18]. As in our case, no evidence for a direct cooperative interaction of the assisting transcription factors with the Hox protein was detected. Thus, we postulate that the Hox-dependent regulation of rpr expression is, as in the case of sal repression by Ubx, achieved through combinatorial regulation, in which two or more regulatory proteins bind to nearby sites, but not necessarily to each other[18]. While combinatorial regulation of gene expression has been extensively studied for diverse transcription factors[52],[53], our results shed new light on the mechanisms of Hox target gene regulation. Previously, much attention focused on the Hox cofactors Extradenticle( Exd) and Homothorax( Hth), which allow Hox proteins to differentially recognize and select some of their target genes through cooperative complex formation[54],[55],[56],[57]. Although Hox cofactors like Exd can explain why different Hox proteins have different DNA binding specificities, the interaction with these factors was not able to explain how broadly expressed Hox proteins are able to affect target gene expression in only a subset of cells. One of the major reasons for that is that Exd and Hth, the only well-known Hox cofactors, are expressed throughout the embryo and interact promiscuously with most Hox proteins. In addition, studies on Exd and Hth revealed that Hox target gene regulation more or less inevitably includes complex formation between Hox proteins and assisting co-regulatory factors. We and others have now shown that the ability of a broadly expressed Hox protein to regulate a target gene in a proper spatial and temporal context is achieved by the Hox-independent recruitment of context-specific transcription factors to cis-regulatory sequences present in compact HREs[18]. Based on our findings, we now suggest that a large number of transcription factors could dictate the transcriptional output in combination with the respective Hox protein by binding selectively and independently to cis-regulatory sequences within HREs of target genes( Figure 7I). Since every cell has a unique combination of transcription factors, the combinatorial interactions for the broadly expressed Hox proteins are almost limitless in such a scenario, accounting for the precise modulation and fine-tuning of Hox target gene regulation, even on the level of the individual cell. Additionally, this model can explain how Hox proteins can act as repressors in one context and as activators in another, because the combined transcriptional output is dependent on the regulatory activity of all transcription factors assembled on a HRE( Figure 7I). There are several lines of evidence that support this model: first, the invariable ectopic activation of Hox downstream genes in spatially restricted domains of every segment when upstream Hox proteins are ubiquitously mis-expressed[8], and second, the accumulation of binding sites for additional transcription factors in enhancers predicted to be controlled by Hox proteins[8]. Alternatively, it seems also possible that Dfd regulates the expression of its target gene rpr only in some maxillary cells, while the novel co-regulatory factors mediate regulation in other maxillary cells. However, since Dfd protein is present in all cells of the segment, the first model seems more likely. Irrespective of the mechanism used by Dfd, it will be essential to study the architecture of HREs, with a special focus on the binding site composition of these enhancers and the diverse factors binding to them to further advance our understanding of Hox target gene regulation in vivo. It has been argued before that context-specific transcription factors assisting Hox proteins in target gene regulation are not likely to act as transcriptional repressors or activators themselves, but rather recruit co-repressors and/or co-activators, and thereby dictate the transcriptional output imprinted in HREs[18],[58]. Our finding of the co-repressor Groucho playing a role in the Dfd-dependent repression of rpr transcription now substantiates this hypothesis, since three of the factors identified in our work, En, Slp1 and Brk, are known to require interactions with the Groucho co-repressor for the transcriptional repression of some of their downstream genes[24],[25],[59]. Interestingly, at least two other co-regulatory transcription factors identified in this work are also known to interact with co-activators/co-repressors: Apt is able to recruit the transcriptional co-activator Multiprotein bridging factor 1( MBF1), thereby mediating Apt-dependent transcriptional activation[35]. Disco has been found in a yeast-two-hybrid screen to interact with the well-known co-repressor C-terminal Binding Protein( CtBP)[60], which is also recruited by Brk to repress some Dpp-responsive genes[24]. Since there is accumulating evidence that co-repressors, like CtBP, execute their function on transcriptional regulation through chromatin modification[61],[62], it is tempting to speculate that Hox proteins regulate their target genes also by epigenetic control mechanisms. In summary, the multitude of potential regulatory mechanisms used by Hox proteins might be the reason why it has been impossible to fully elucidate how Hox proteins mediate their function with high specificity and precision in vivo. In the light of recent advances, it now seems likely that the mechanisms leading to functional specificity of Hox proteins are dependent on the cellular context, the composition of the target enhancer element and the identity of the individual Hox protein. Thus, one could argue that during evolution Hox proteins have undergone individualization in trans, as well as sequence diversification in cis.
Bilateral animals share a common genetic mechanism to control development along the anterior–posterior body axis , and transcription factors of the Hox class are key regulators of this conserved process . It is thought that Hox proteins drive morphological diversification of body segments by differentially controlling the expression of downstream genes . However , due to their highly conserved DNA binding domain , the homeodomain , Hox proteins alone bind to very similar and frequently occurring sequences in the genome . This implies that Hox proteins alone are likely insufficient to activate or repress their target genes in a proper spatio-temporal fashion . In contrast to this observation , Hox proteins have very specific effects during development and execute their function with high precision . A solution to this paradox could lie in the context-specific interaction of Hox proteins with other transcriptional regulators; however , only a few examples are known . By analysing the mechanism underlying the regulation of the Hox target gene reaper , we identified a set of eight transcription factors to be important for the precise spatio-temporal regulation of this gene . Based on our findings , we suggest that Hox proteins functionally interact with a plethora of unrelated transcription factors on small , yet complex enhancer elements to execute their specific functions throughout development of diverse organisms .