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Title: Regulation of the BMP Signaling-Responsive Transcriptional Network in the Drosophila Embryo}
Article:
Abstract:
The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.
Introduction:
The Bone Morphogenetic Protein( BMP) signaling pathway is used repeatedly throughout development to regulate a diverse array of processes. One of the major conserved roles of the BMP pathway is to specify epidermal fates during embryonic dorsal-ventral axis patterning[1]. For example, in Drosophila, a heterodimer of the BMP signaling molecules, Decapentaplegic( Dpp) and Screw( Scw), forms a gradient in order to pattern the dorsal ectoderm of the embryo into dorsal epidermis and amnioserosa fates. Dpp-Scw signaling through a Thickveins( Tkv), Punt and Saxophone receptor complex results in phosphorylation of the C-terminal tail of the Mothers Against Dpp( Mad) transcription factor( pMad), which then interacts with Medea( Med), leading to their stabilization in the nucleus[2]. Initially, nuclear pMad is observed around the dorsal midline following extracellular transport of the Dpp-Scw ligand as the first step in gradient formation[2]. The pMad distribution is then refined to a narrower stripe by positive feedback via transcription of Dpp-Scw target genes, including that of eiger( egr), which encodes a TNF-α ligand[3]. Studies in flies and vertebrates have revealed complex regulation of Smad transcription factors by phosphorylation, dephosphorylation, SUMOylation and ubiquitination, which controls their subcellular localization, transcriptional activity and degradation[1, 4]. Mad and Med both activate and repress transcription[5]. Smad-dependent activation can be attenuated by the Brinker( Brk) repressor, which is expressed in the neuroectoderm underlying the dorsal ectoderm[6]. Evidence has been obtained for competitive binding between Mad and Brk at Dpp-responsive enhancers during different developmental stages[5]. In terms of Mad-Med activation, DNA binding motifs have been identified from which loose consensus sequences have been derived[7]. In addition, a well-characterized, conserved Activation Element has been described, along with a related Silencer Element that mediates Dpp-dependent repression via recruitment of the Schnurri( Shn) corepressor[5]. Genomics approaches have facilitated identification of developmental gene networks in the Drosophila embryo, particularly those activated by the Dorsal transcription factor or underpinning mesoderm patterning in the embryo[8, 9]. Despite progress in deciphering the Dorsal gene regulatory network during early patterning of the embryo, details relating to the transcription network underpinning dorsal ectoderm differentiation in response to Dpp are sparse. In this study, we investigate the transcriptional responses downstream of the major developmental BMP signaling pathway in the Drosophila embryo. Our data identify multiple target genes and enhancers, and reveal that the EGF pathway activity is constrained in the embryo to facilitate correct Dpp-dependent patterning. In addition, we show roles for Zelda( Zld), Zerknüllt( Zen) and the BEAF-32 insulator protein in Dpp gradient interpretation.
Discussion:
We have combined RNA-seq, ChIP-seq and expression pattern data to identify the Dpp-responsive regulatory network in the early Drosophila embryo. Some of the Dpp target genes we have identified encode proteins with predicted functions compatible with the different events in the life of amnioserosa cells[40, 41], including cell division arrest–Toucan( Toc)[42] and Ana[43], changes in cell shape and adhesion–Bazooka( Baz)[44] and Rst[45], and programmed cell death during dorsal closure–Rst[46] and Scylla( Scyl)[47]. It will be interesting to precisely determine the timings at which these different early Dpp target genes are required. We have also identified new Dpp target genes for which a function is currently lacking. As the Dpp-responsive gene network appears relatively modest, given the tractability of the Drosophila embryo, this network therefore represents a relatively simple framework for future studies aimed at understanding how gene expression changes drive cell fate changes. Many components of the EGF pathway are regulated by Dpp signaling, and our data support a model whereby EGF signaling regulates Dpp transduction through the controlled inhibition of Mad activity( Fig 8A). During embryonic stage 5, rho and vein are activated by Dpp signaling, enabling dorsal MAPK pathway activation[48]. Based on data from vertebrates showing that MAP kinase phosphorylation of Smad1 ultimately represses its activity by leading to its degradation[4], we propose that the activated dpERK promotes pMad degradation in the early embryo. Therefore, in our model the level of pMad is a balance between BMP receptor-mediated phosphorylation and linker phosphorylation initiated by dpERK( Fig 8A). In a wildtype embryo, this equilibrium establishes a nuclear pMad level that activates Dpp target genes including egr, with Egr then functioning via positive feedback to promote refinement of the pMad stripe at the dorsal midline, to allow activation of peak Dpp target genes( Fig 8A). Embryos mutant for kek1, encoding an inhibitor of the EGF receptor[18], have a narrower domain of dpERK activation, which we attribute initially to the EGF ligand being sequestered by a greater number of uninhibited EGF receptors( Fig 8A). This narrower dpERK domain alters the pMad-dpERK equilibrium, so that pMad levels are dampened from stage 5. The lower pMad in kek1 mutant embryos likely reduces expression of the positive target, rho, and leads to loss of egfr repression, which would contribute to the shrinking of the dpERK domain compared to wildtype embryos. As a result, egr activation and therefore positive feedback at stage 6 is compromised, leading to loss of refinement of the pMad stripe, and disrupted target gene expression( Fig 8A). Conversely, in egfr or rho mutant embryos, the loss/reduction in dpERK leads to expanded Race expression, consistent with the pMad-dpERK equilibrium being tipped in favour of higher pMad( Fig 8A). Together, these data suggest that the presence of both positive and negative regulators of EGF signaling renders the BMP network self-regulating, in that it can maintain a threshold of EGF signaling that is permissive for Dpp-dependent patterning. This Dpp-EGF interplay may also be important in other developmental contexts, such as the dorsal head region in the Drosophila embryo where overactivation of EGF signaling antagonises Dpp signaling[49]. Our ChIP data has allowed the identification of many Dpp dependent enhancers, with integration of our ChIP- and RNA-seq data suggesting that the majority of Dpp-responsive genes in the early embryo are direct targets. We have also shown that ~20% of the pMad/Brk genomic regions are bound by Dorsal, Twist and Snail, suggesting that the enhancers of some positive pMad targets are inactive in the mesoderm and neuroectoderm, not only due to Dorsal repression of the dpp gene, but also due to direct repression by Dorsal and/or Snail binding. These targets, including transcription factors and signaling pathway components, might be potent in transforming cell fate so that it is advantageous to have them under an additional level of negative control in the mesoderm and neuroectoderm. In addition, some dual Dpp/Dorsal targets include genes expressed in the neuroectoderm that may be activated by Dorsal in the neuroectoderm and repressed by Dpp signaling in the dorsal ectoderm, as described for brk[6]. Most of the pMad genomic regions we have identified are also bound by Zld and we show that Zld is required for Race enhancer activity, with extra Zld sites broadening the expression pattern. This latter effect may be through increased chromatin accessibility, as described for Dorsal activation[50]. Our data also show that Zen is important for peak and intermediate Dpp target gene expression, consistent with that reported for Race, C15, egr and cv-2 previously[3, 21, 22]. zen is initially broadly expressed in the dorsal ectoderm but becomes restricted to the dorsal midline in response to Dpp signaling[15]. Given that tup and ush have broader expression patterns than the restricted zen pattern, it appears that the Zen prepattern is important for expression of at least some Dpp target genes. We also found that insulator proteins bind to many of the pMad/Brk ChIP enhancers, including those for Race, ana, egr, ush, Ama and pnr. Consistent with this, a set of enhancers enriched for mesodermal enhancers has recently also been reported to show significant overlap with insulator proteins[51]. We also found insulator binding to other early embryonic enhancers( Zld, Dorsal), but to a lesser extent for a set of enhancers that are activated throughout embryogenesis, suggesting that insulator binding may be important for early activation. Analysis of BEAF-32 null embryos showed a small but significant reduction in amnioserosa cell number and disrupted Race activation. This relatively mild phenotype, and the viability of the BEAF-32 null embryos, could reflect redundancy in insulator binding proteins. Recently, Drosophila Mad and Med have been found to colocalize with CTCF in tissue culture cells in the absence of Dpp signaling, but relocalize away from these regions when Dpp is present[52]. Consistent with this, we detect very poor overlap between regions bound by CTCF and the signaling activated form of Mad. Insulators can form gene loops with active promoters in Drosophila[53, 54] and for some insulator binding proteins, including BEAF-32 and CP190, ChIP-seq has identified indirect binding peaks that have been attributed to long-range interactions. These include insulator-promoter interactions, with GAF-regulated paused RNA polymerase II( Pol II) at the promoter proposed to act as bait for the insulator interaction[55]. Such interactions may explain why we observe NELF binding to a subset of the pMad/Brk genomic regions. In addition, enhancer-promoter loops have been found to be developmentally stable and associated with Pol II pausing[51]. Based on our data and the findings described above, we propose a speculative model for Dpp-dependent activation whereby binding of the Zld pioneer transcription factor opens chromatin to allow recruitment of other proteins, including Zen( with the early broad expression important) and insulator proteins( Fig 8B). Insulator binding may promote looping of the enhancer to the promoter, with the majority of Dpp target genes harbouring paused Pol II[56, 57]. Subsequent recruitment of pMad, potentially aided by an interaction with Zen[22] and/or insulator proteins, would allow release of paused Pol II and gene activation. Parallels exist here with vertebrate Smads, as a potential role for pioneer transcription factors in Smad-dependent activation has been proposed[58]. In addition, Smad3 is recruited to the H19 Imprinting control region by CTCF, via a direct interaction shown between Smad3 and CTCF[59], suggesting that insulator proteins may also play a role in Smad-dependent activation in vertebrates.
Embryogenesis involves the patterning of many different cell fates by a limited number of types of signals . One way that these signals promote a particular cell fate is through the induction of a complex , yet highly reproducible , gene expression programme that instructs changes in the cell . For example , there is a conserved role for BMP signals in specifying cell fates during dorsal-ventral axis patterning . Here , we have used genomics approaches to identify the gene expression programme implemented in response to BMP signaling during axis patterning in the Drosophila embryo . Part of the gene network downstream of BMP signaling includes members of the EGF signaling pathway , with our data highlighting reciprocal interactions between these two pathways . We have also determined genome-wide binding of BMP-responsive transcription factors to gain new insights into how the BMP gene network is activated . Our data reveal roles for specific transcription factors and insulator binding proteins , with the latter traditionally associated with the separation of transcriptional domains . Overall , our data will provide a platform for exploiting the tractability of the Drosophila embryo to determine which features of the network are critical drivers of BMP-induced cell fate changes during embryogenesis .