dorsal : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - dorsal

Synonyms -

Cytological map position - 36C2-9

Function - transcription factor

Keywords - morphogen, DV polarity, dorsal group, immune response, Dorsal and DIF act downstream of the Toll pathway

Symbol - dl

FlyBase ID:FBgn0260632

Genetic map position - 2-52.9

Classification - rel homolog

Cellular location - nuclear and cytoplasmic

NCBI link: Entrez Gene
dl orthologs: Biolitmine
Recent literature
Chowdhury, M., Zhang, J., Xu, X. X., He, Z., Lu, Y., Liu, X. S., Wang, Y. F. and Yu, X. Q. (2019). An in vitro study of NF-kappaB factors cooperatively in regulation of Drosophila melanogaster antimicrobial peptide genes. Dev Comp Immunol 95: 50-58. PubMed ID: 30735676
An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-kappaB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IkappaB protein Cactus, while Relish contains an IkappaB-like inhibitory domain at the C-terminus. NF-kappaB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-kappaB homodimers and heterodimers are not well understood. This study compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. The nucleotides at the 6th and 8th positions of the 3' half-sites of the kappaB motifs were identified that are important for the specificity and activity of NF-kappaB transcription factors.
Bandodkar, P. U., Al Asafen, H. and Reeves, G. T. (2020). Spatiotemporal control of gene expression boundaries using a feedforward loop. Dev Dyn. PubMed ID: 31925874
A feedforward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen-mediated tissue patterning. However, most morphogen gradients also evolve in time. The spatiotemporal behavior of a coherent FFL in two contexts was studied: (a) a generic, oscillating morphogen gradient and (b) the dorsal-ventral patterning of the early Drosophila embryo by a gradient of the NF-kappaB homolog Dorsal with its early target Twist. In both models, features in the dynamics of the intermediate node-phase difference and noise filtering-that were found to be largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the Dorsal gradient model, it was also found that proper target gene expression was not possible without including the effect of maternal pioneer factor Zelda. gAn FFL buffers fluctuation to changes in the morphogen signal ensuring stable gene expression boundaries.
Al Asafen, H., Bandodkar, P. U., Carrell-Noel, S., Schloop, A. E., Friedman, J. and Reeves, G. T. (2020). Robustness of the Dorsal morphogen gradient with respect to morphogen dosage. PLoS Comput Biol 16(4): e1007750. PubMed ID: 32251432
In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. This work investigated the robustness of the Dorsal/NF-kappaB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and an empirical description of the Dorsal gradient was found to be highly sensitive to maternal dorsal dosage. In contrast, it was found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, a mechanistic model of the Dorsal signaling module was analyzed, and Cactus, a cytoplasmic inhibitor for Dorsal, had to be be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. This work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.
Keller, S. H., Jena, S. G., Yamazaki, Y. and Lim, B. (2020). Regulation of spatiotemporal limits of developmental gene expression via enhancer grammar. Proc Natl Acad Sci U S A 117(26): 15096-15103. PubMed ID: 32541043
The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, this study measured the contribution of individual binding sites in transcriptional regulation. Binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, this study shows that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. It is proposed that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.
Li, R., Yao, X., Zhou, H., Jin, P. and Ma, F. (2021). The Drosophila miR-959-962 Cluster Members Repress Toll Signaling to Regulate Antibacterial Defense during Bacterial Infection. Int J Mol Sci 22(2). PubMed ID: 33477373
MicroRNAs (miRNAs) are a class of ~22 nt non-coding RNA molecules in metazoans capable of down-regulating target gene expression by binding to the complementary sites in the mRNA transcripts. Many individual miRNAs are implicated in a broad range of biological pathways, but functional characterization of miRNA clusters in concert is limited. This study reports that miR-959-962 cluster (miR-959/960/961/962) can weaken Drosophila immune response to bacterial infection evidenced by the reduced expression of antimicrobial peptide Drosomycin (Drs) and short survival within 24 h upon infection. Each of the four miRNA members is confirmed to contribute to the reduced Drs expression and survival rate of Drosophila. Mechanically, RT-qPCR and Dual-luciferase reporter assay verify that tube and dorsal (dl) mRNAs, key components of Toll pathway, can simultaneously be targeted by miR-959 and miR-960, miR-961, and miR-962, respectively. Furthermore, miR-962 can even directly target to the 3' untranslated region (UTR) of Toll. In addition, the dynamic expression pattern analysis in wild-type flies reveals that four miRNA members play important functions in Drosophila immune homeostasis restoration at the late stage of Micrococcus luteus (M. luteus) infection. Taken together, these results identify four miRNA members from miR-959-962 cluster as novel suppressors of Toll signaling and enrich the repertoire of immune-modulating miRNA in Drosophila.
Zhou, H., Li, S., Wu, S., Jin, P. and Ma, F. (2021). LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis. Int J Mol Sci 22(18). PubMed ID: 34576280
Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. Firstly, this study discovered that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, the results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, the findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w(1118)) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, this study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity.
Carmon, S., Jonas, F., Barkai, N., Schejter, E. D. and Shilo, B. Z. (2021). Generation and timing of graded responses to morphogen gradients. Development 148(24). PubMed ID: 34918740
Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. This study examined whether morphogens can also induce a graded response within such domains. To this end, the role was explored. of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.
Hegde, S., Sreejan, A., Gadgil, C. J. and Ratnaparkhi, G. S. (2022). SUMOylation of Dorsal attenuates Toll/NF-kappaB signaling.. Genetics 221(3). PubMed ID: 35567478
In Drosophila, Toll/NF-kappaB signaling plays key roles in both animal development and in host defense. The activation, intensity, and kinetics of Toll signaling are regulated by posttranslational modifications such as phosphorylation, SUMOylation, or ubiquitination that target multiple proteins in the Toll/NF-kappaB cascade. This study has generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant. Intriguingly, embryos laid by dlSCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DLSCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dlSCR animals show an increase in crystal cell numbers, stronger activation of humoral defense genes, and high cactus levels. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (1-5%) suggests that it acts to block transcriptional activation, which is driven primarily by DL that is not SUMO conjugated. These findings define SUMO conjugation as an important regulator of the Toll signaling cascade, in both development and host defense. These results broadly suggest that SUMO attenuates DL at the level of transcriptional activation. Furthermore, it is hypothesized that SUMO conjugation of DL may be part of a Ubc9-dependent mechanism that restrains Toll/NF-kappaB signaling.
Lusk, J. B., Chua, E. H. Z., Kaur, P., Sung, I. C. H., Lim, W. K., Lam, V. Y. M., Harmston, N. and Tolwinski, N. S. (2022). A non-canonical Raf function is required for dorsal-ventral patterning during Drosophila embryogenesis. Sci Rep 12(1): 7684. PubMed ID: 35538124
Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. This study describes a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. These results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.
Chahda, J. S., Ambrosi, P. and Mizutani, C. M. (2022). The nested embryonic dorsal domains of BMP-target genes are not scaled to size during the evolution of Drosophila species. J Exp Zool B Mol Dev Evol. PubMed ID: 35451554
Egg size is a fast-evolving trait among Drosophilids expected to change the spatial distribution of morphogens that pattern the embryonic axes. This study asked whether the patterning of the dorsal region of the embryo by the Decapentaplegic/Bone Morphogenetic Protein-4 (DPP/BMP-4) gradient is scaled among Drosophila species with different egg sizes. This region specifies the extra-embryonic tissue amnioserosa and the ectoderm. This study found that the entire dorsal region scales with embryo size, but the gene expression patterns regulated by DPP are not proportional, suggesting that the DPP gradient is differentially scaled during evolution. To further test whether the DPP gradient can scale or not in Drosophila melanogaster, embryos were created with expanded dorsal regions that mimic changes in scale seen in other species, and the resulting domains were measured of DPP-target genes. The proportions of these domains were found to not be maintained, suggesting that the DPP gradient is unable to scale in the embryo. These and previous findings suggest that embryonic dorso-ventral patterning lacks scaling in the ventral and dorsal sides but is robust in the lateral region where the neuroectoderm is specified and two opposing gradients, Dorsal/NFkappa-B and DPP, intersect. It is proposed that the lack of scaling of the DPP gradient may contribute to changes in the size of the amnioserosa and the number of ectodermal cells with specific cortical tensions, which are expected to generate distinct mechanical forces for gastrulating embryos of different sizes.
Syed, S., Duan, Y. and Lim, B. (2023). Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos. bioRxiv. PubMed ID: 36711729
It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. This study utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, it was found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in total mRNA production of the target gene. Evidence is provided of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. It is proposed that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.
Cai, Q., Guo, H., Fang, R., Hua, Y., Zhu, Y., Zheng, X., Yan, J., Wang, J., Hu, Y., Zhang, C., Zhang, C., Duan, R., Kong, F., Zhang, S., Chen, D. and Ji, S. (2022). A Toll-dependent Bre1/Rad6-cact feedback loop in controlling host innate immune response. Cell Rep 41(11): 111795. PubMed ID: 36516751
The Toll signaling pathway was initially identified for its involvement in the control of early embryogenesis. It was later shown to be also part of a major innate immune pathway controlling the expression of anti-microbial peptides in many eukaryotes including humans; cactus, the essential negative regulator of this pathway in flies, was found to be induced in parallel to the Toll-dependent activation process during immune defenses. This study was interested in the mechanisms of this dual effect and provides evidence that upon pathogenic stimuli, Dorsal, one of the transcription factors of the fly Toll pathway, can induce the expression of the E3 ligase Bre1. It was further shown that Bre1 complexes with the E2 Rad6 to mono-ubiquitinate histone H2B and to promote the transcription of cactus to achieve homeostasis of the Toll immune response. These studies characterize a Toll signal-dependent regulatory machinery in governing the Toll pathway in Drosophila.
Alamos, S., Reimer, A., Westrum, C., Turner, M. A., Talledo, P., Zhao, J., Luu, E. and Garcia, H. G. (2023). Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient. Cell Syst. PubMed ID: 36696901
How enhancers interpret morphogen gradients to generate gene expression patterns is a central question in developmental biology. Recent studies have proposed that enhancers can dictate whether, when, and at what rate promoters engage in transcription, but the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, this study established a minimal promoter-proximal synthetic enhancer in embryos of Drosophila melanogaster. A gradient of the Dorsal activator was found to be read by a single Dorsal DNA binding site. Using live imaging to quantify transcriptional activity, it was found that a single binding site can regulate whether promoters engage in transcription in a concentration-dependent manner. By modulating the binding-site affinity, it was determined that a gene's decision to transcribe and its transcriptional onset time can be explained by a simple model where the promoter traverses multiple kinetic barriers before transcription can ensue.


Dorsal (DL) is the focal protein in the development of dorsoventral polarity in the developing fly. It is a transcription factor, activating and repressing zygotic genes responsible for differentiation along the dorsoventral axis during the early stages of development. Over the course of time measured in hours, even before cellularization, the rapidly dividing zygotic cells take up their positions at the periphery of the fertilized egg. Nuclei in the ventral portion stain positive for DL while nuclei in the dorsal region fail to stain. Nuclear staining is graded; nuclei closest to the ventral midline stain most strongly (Rushlow, 1989, Steward, 1989 and Roth, 1989).

The dorsal group comprises a whole class of genes acting in the mother and responsible for setting up the activation and nuclear transport of DL. Early in oogenesis the egg becomes polarized. Such polarization affects the follicle cells surrounding the egg. Ventral follicle cells take on a different developmental fate from dorsal follicle cells. For example, the release and activation of the Toll ligand Spätzle occurs only in the ventral region of the egg.

Once fertilization has taken place, Spätzle triggers signaling in the Toll receptor. The signals are transmitted via Tube and Pelle into the cytoplasm, resulting in the activation of Dorsal. Activation of Dorsal is held in abeyance by Cactus protein. Cactus binds Dorsal, preventing its nuclear transport. Signals from Toll result in the destruction of Cactus in ventral cells, whereupon Dorsal is released to continue its transportation into the nucleus.

The Cactus-Dorsal interactive system is preserved in vertebrates and used in activation of the immune system. The transcription factor NF kappa B is involved in the activation of immune system cells. The vertebrate homolog of Cactus, I kappa B (where I stands for inhibitor), keeps NF kappa B restrained in the cytoplasm until cell activation, at which time NF kappa B enters the nucleus. The adult fly uses the identical system in its immune system. Bacterial challenge causes the activation of Dorsal and a second Dorsal-like factor (Dif) in the fat body, an organ that serves the immune function in flies. The developing awareness of the importance of an immune response system in flies has initiated a new focus of interest in Dorsal research (Reichart, 1993 and Lemaitre, 1995).

Dorsal's nuclear function involves an interaction with transcription factor Bang senseless, here termed Dorsal switch protein 1 or DSP1 (FlyBase link: FBgn0000231) DSP1, an HMG-1/2-like protein, binds DNA in a highly cooperative manner with three members of the Rel family of transcriptional regulators (NF-kappaB, the p50 subunit of NF-kappaB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. In HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the twist promoter and inhibits that activation from the zen promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion (Brickman, 1999).

DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman, 1999).

It is not known what DNA sequences in the zen and twist promoters determine the opposite effects of DSP1 on dorsal-mediated activation. The finding that a negative regulatory element (NRE) has no effect on cooperative binding to DNA of DSP1 and various Rel proteins prompted a reexamination of the earlier claims that DSP1 converts Dorsal, the p50 homodimer, and the NF-kappaB heterodimer into repressors and that this effect requires the NRE. In each case, DSP1 inhibits Rel-protein-dependent activation both in the presence and absence of an NRE. In no case was NRE-dependent conversion of the Rel protein to a repressor by cotransfection with DSP1 observed. It is not understood why the current results differ from those reported previously (Brickman, 1999 and references therein).

Sites of the described protein-protein interactions are found in the conserved Rel domains and in the fragment of DSP1 that bears both HMG domains. The Rel domains of p65 and of Dif differ from those of Dorsal and of p50 in that they lack the HMG-domain-interaction site. The HMG domain of DSP1 also interacts with the TATA-binding protein. Similar interactions have been reported for HMG-1 and HMG-2 with the steroid hormone receptors, for HMG-1 with p53, for HMG-1 with HOXD9, and for HMG-2 with Oct2. Thus, the HMG domain may contain a common structural motif for cooperative DNA binding and interaction with other transcription factors. The interaction between TATA-binding protein and DSP1 also seems to be influenced by the glutamine-rich amino-terminal domain in that the full-length DSP1 interacts more avidly with TATA-binding protein than does the HMG-1 domain. These experiments suggest that the amino-terminal glutamine-rich domain may also potentiate the DSP1-Rel protein interaction as well, because all DSP1-Rel interactions seem stronger with full-length DSP1, particularly the weak interactions seen between DSP1 and p65 or Dif, which are observed only with GST-DSP1 and not with GST-DSP1 (178-393) (Brickman, 1999).

Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo

Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. This study determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. This analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development (Zeitlinger, 2007).

ChIP-chip assays were performed with antibodies directed against Dorsal, Twist, or Snail on Toll10b mutant embryos, aged 2-4 h. These embryos contain a constitutively activated form of the Toll receptor, which results in high levels of nuclear Dorsal protein and uniform expression of Twist and Snail throughout the embryo. The high levels of Dorsal, Twist, and Snail cause all cells to form derivatives of the mesoderm at the expense of neurogenic and dorsal ectoderm. Thus, these embryos represent a uniform cell type with respect to DV fate (Zeitlinger, 2007).

The whole-genome ChIP-chip experiments reveal several hundred strong binding clusters of Dorsal, Twist, and Snail with up to 40-fold ChIP enrichment, most of which span regions of ~1 kb in length. To identify the binding patterns of bona fide target enhancers of the Dorsal regulatory network, known enhancers were analyzed. The 22 known enhancers fall into three classes: type 1, type 2, and type 3, based on which levels of nuclear Dorsal regulate their expression (Zeitlinger, 2007).

The 10 type 1 enhancers (associated with twi, sna, miR-1, htl, hbr, mes3, CG12177, ady43A, tin, and Phm) are activated by peak levels of Dorsal in the presumptive mesoderm, and are all constitutively activated in Toll10B mutant embryos. The ChIP-chip experiments identify strong binding peaks (greater than fivefold enrichment) of Dorsal, Twist, and Snail (DTS) within five of the 10 enhancers (twi, sna, miR-1, CG12177 and Phm). Another three enhancers, those associated with htl, tin, and ady43A, show significant but lower (less than fivefold) binding peaks restricted to Twist and Snail (TS) binding. This observation is consistent with earlier studies indicating that these enhancers might be primarily activated by Twist. Hence, eight of the 10 known type 1 enhancers exhibit significant in vivo occupancy by Twist and Snail (Zeitlinger, 2007).

An even greater correspondence between known enhancers and in vivo occupancy is seen for the type 2 [sim, E(spl), vn, rho, vnd and brk] and type 3 enhancers (ths, sog, ind, dpp, zen and tld), which are regulated by intermediate and low levels of the Dorsal gradient, respectively. All 12 enhancers are silenced in Toll10B mutant embryos due to constitutive expression of the Snail repressor. Remarkably, every enhancer exhibits strong DTS or TS peaks with greater than fivefold enrichment in the ChIP-chip assays. Thus, ChIP-chip assays correctly identified 20 of the 22 known Dorsal target enhancers (Zeitlinger, 2007).

Most known DV enhancers are associated with overlapping binding clusters of Dorsal, Twist, and Snail regardless of whether they mediate activation or repression. Moreover, 17 of the 20 binding clusters at known enhancers display greater than fivefold enrichment of Twist and/or Snail. Using these binding criteria, 428 high-confidence DTS regions and 433 high-confidence TS regions were identified across the genome (Zeitlinger, 2007).

To confirm these regions through independent evidence, sequence analysis on these regions was performed using the known consensus binding motifs of Dorsal, Twist, and Snail. As expected, the identified regions are highly enriched in all three binding motifs. Moreover, a large fraction of the motifs is conserved across the 12 sequenced Drosophila species providing evidence that the discovered regions are functionally important. Finally, when motifs that are enriched in these regions were identified de novo, the known binding motifs can be rediscovered. Hence, the regions identified represent putative target gene enhancers of the DV network (Zeitlinger, 2007).

To show that newly identified regions indeed function as enhancers in vivo, putative enhancers were selected of primary DV genes; i.e., those genes that are expressed as localized stripes across the DV axis. In addition to the 22 known DV enhancers, 47 new putative enhancers were identified , some of which appear to regulate the same gene, were identified. By attaching the genomic sequence to a lacZ reporter and expressing the construct in transgenic embryos, seven of these enhancers were shown to be bona fide DV enhancers and that regulation by multiple enhancers occurs (Zeitlinger, 2007).

The wntD gene is expressed in portions of the presumptive mesoderm where it mediates feedback inhibition of Toll signaling. A cluster of DTS-binding peaks was identified in the 5'-flanking region, and the corresponding genomic DNA fragment mediates lacZ expression in the same region of the mesoderm as the endogenous gene. Similar results were obtained with the DTS-binding cluster located in the 5'-flanking region of mes5/mdr49 (Zeitlinger, 2007).

The vnd locus contains a well-documented intronic enhancer that mediates expression in the neurogenic ectoderm and recapitulates the spatial and temporal expression pattern of the endogenous gene. The ChIP-chip analysis detected this enhancer but also revealed two novel clusters further upstream. When tested for lacZ reporter activity, these novel genomic sequences directed lacZ expression in a pattern resembling that of the endogenous gene over different time periods: One directs early vnd expression in the presumptive ventral neurogenic ectoderm (vNE) while the other directs later expression in the medial column (mc) of the developing nervous system. All three enhancers contain evolutionarily conserved binding sites for Dorsal, Twist, and Snail, suggesting that the enhancers are not redundant but may function to fine-tune the vnd expression pattern. Overlapping enhancer activity was also observed for multiple miR-1 enhancers. Overall, as many as a third of all DV genes have multiple binding clusters, and thus might be subject to similar regulatory control (Zeitlinger, 2007).

Several of the occupied regions are associated with Dpp target genes expressed in the dorsal ectoderm. When the tup and pnr intronic sequences are tested in transgenic embryos, both fragments function as authentic enhancers and direct localized expression in the dorsal ectoderm, comparable to the endogenous tup and pnr expression patterns. These results suggest that the Dorsal patterning network directly regulates the expression of Dpp target genes (see below) (Zeitlinger, 2007).

It was noticed that many of the new DTS/TS clusters are associated with AP genes involved in segmentation. Although classical genetic studies argue that AP and DV patterning of the early embryo are controlled by separate maternal genetic programs, it is conceivable that the expression of AP target genes is modulated by the DV network. Indeed, DV modulation of segmentation gene expression has been observed previously (Zeitlinger, 2007).

The gap gene orthodenticle (otd) is expressed in two stripes across the AP axis in the early embryo. The anterior stripe shows diminished expression on the ventral side. Previous studies identified a 5' enhancer that recapitulates the normal expression pattern, including Dorsal-dependent suppression in ventral regions. ChIP-chip identified a strong DTS cluster within the limits of this enhancer. A similar DV bias in the expression pattern was found for the gap gene tailless (tll) and the pair-rule genes runt and hairy. In each case, the regions identified by ChIP-chip overlap or map close to known regulatory regions and contain several Dorsal-binding motifs (Zeitlinger, 2007).

At the gap gene knirps, a DTS-binding cluster was found in a region distinct from the known Bicoid-dependent enhancer. This newly identified genomic region functions as a bona fide enhancer directing expression in the anteroventral domain like endogenous knirps. Thus, the ChIP-chip analysis identified novel AP regulatory regions modulated by DV activity (Zeitlinger, 2007).

In summary, many segmentation genes contain DTS/TS-binding clusters, and at least some of these regions modulate gene expression across the DV axis, particularly in anterior regions of the embryo. It is concluded that the Dorsal gradient does not only regulate primary DV target genes, but rather appears to fine-tune a large number of genes that do not contribute to DV axis formation themselves, at least based on their known genetic function (Zeitlinger, 2007).

Many DTS/TS-binding clusters are also found at genes encoding signal transduction components. Analysis of the network formed by these pathways suggests that the Dorsal gradient controls the expression of many target genes by multiple regulatory pathways (Zeitlinger, 2007).

Dorsal directly represses Dpp expression in the mesoderm and neuroectoderm, leading to localized Dpp signaling in the dorsal ectoderm. Dpp activates a variety of genes, including tup and pnr. Accurate identification of intronic tup and pnr enhancers suggests that these genes are directly regulated by the Snail repressor, in addition to indirect regulation by the Dorsal gradient via Dpp signaling. zen is another well-known target gene of Dorsal in the dorsal ectoderm, and its product, a homeodomain transcription factor, functions synergistically with Dpp signaling. Target genes of Zen also appear to be subject to additional regulation by the Dorsal gradient. In the dorsal ectoderm, Dorsal may regulate gene expression by two mechanisms: direct repression, and indirect repression via Snail (Zeitlinger, 2007).

Similar network configurations regulate gene expression in the neuroectoderm. High levels of Dorsal repress the expression of rho via Snail in the mesoderm, thereby blocking EGF signaling in Toll10b mutant embryos. ChIP-chip data suggest that the Dorsal network regulates additional genes encoding EGF signaling components as well as EGF target genes such as pnt, aop/yan, and argos. In the case of Notch signaling, it is known that the Dorsal network represses Notch target genes such as sim in Toll10B mutant embryos through Snail. The Dorsal network may also regulate Notch signaling more directly, by suppressing genes encoding components of the signaling pathway including Notch itself (Zeitlinger, 2007).

Although repression of neuroectodermal target genes is likely to occur predominantly through Snail, Dorsal also induces the expression of a number of microRNAs in Toll10b mutant embryos, including miR-1. Some of the neuroectodermal genes repressed by Snail are also predicted targets of these microRNAs. Hence, there may be multiple tiers of repression in the DV system, similar to the activities of the gap repressors in the AP system (Zeitlinger, 2007).

In summary, the present ChIP-chip study revealed an unexpectedly broad distribution of binding peaks for Dorsal, Twist, and Snail in the genome, and suggests extensive integration of the Dorsal regulatory network with additional patterning processes, such as Dpp signaling in the dorsal ectoderm and segmentation across the AP axis. In addition to the observed tight correlation between binding peaks and known enhancers, two lines of evidence suggest that a significant fraction of the newly identified regions is functional: First, the bound regions are highly enriched in evolutionarily conserved Dorsal, Twist, and Snail sequence motifs; and, second, several of the identified enhancers were experimentally confirmed by lacZ reporter gene expression in transgenic embryos. Thus, while genetic studies identified core sets of regulators for each developmental process in Drosophila, gene regulation integrates information more widely from several different systems. It is likely that integration of diverse patterning processes will also apply to mammalian development, including stem cell differentiation (Zeitlinger, 2007).

Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal (DL) Rel homology domain. This study shows that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters (Ratnaparkhi, 2009).

The Drosophila genome encodes three rel homology domain (RHD) containing proteins, Dorsal (Dl), Dorsal-related immunity factor (Dif), and Relish (Rel). The RHD, which is also found in the human NFκB family of transcriptional activators, mediates dimerization and sequence-specific DNA binding. Rel/NFκB family proteins in vertebrates and invertebrates play central roles in the innate immune response by triggering the expression of antimicrobial defense genes in response to signals transduced by Toll and the Immune deficiency (Imd) signal transduction pathways. In Drosophila, Dl also directs dorsoventral (D/V) patterning of the embryo. Specifically, the regulated nuclear localization of maternally expressed Dl in response to Toll signaling in the embryo leads to the formation of a ventral-to-dorsal nuclear concentration gradient of Dl and to the spatially restricted regulation of a large number of genes, including twist (twi), snail (sna), and rhomboid (rho), which are activated by Dl, and zerknullt and decapentaplegic, which are repressed by Dl. This serves to subdivide the embryo into multiple developmental domains along its D/V axis (Ratnaparkhi, 2009).

Unlike Dl, Dif and Rel are not required for D/V patterning. Instead, these two rel-family proteins function along with Dl in the innate immune response. Toll signaling in the immune system leads to the translocation of Dl and Dif to the nucleus and the consequent activation of a subset of anti-microbial defense genes, including drosomycin (drs) and Immune induced molecule 1. Dl and Dif are believed to have redundant roles in this process and thus either one alone is sufficient for the induction of drs. Activation of the Imd signal transduction pathway, leads to proteolytic cleavage of Rel. The N-terminal region of Rel, which contains the RHD, then translocates into the nucleus where it activates expression of anti-bacterial genes, such as diptericin (dipt), cecropin-A1 (cec-A), and attacin-A. Dl, Dif, and Rel homo- and hetero-dimerize to activate different subsets of the anti-microbial defense genes in response to signals from the Toll and Imd pathways (Ratnaparkhi, 2009).

Very little is known about the identity of factors that assist the RHD proteins in the activation of the anti-microbial defense genes. Proteins that modulate expression of these genes include transcription factors such as the GATA factor Serpent (Srp), Hox factors, Helicase89B, and an unknown protein that binds region 1 (R1), a regulatory module in cec-A and other anti-microbial defense genes. In addition, a recent screen identified several POU domain proteins as potential regulators of anti-microbial defense genes (Ratnaparkhi, 2009).

To date, about a dozen proteins that interact directly with Dl and modulate its regulatory functions have been identified by genetic and biochemical means. For example, an interaction between Dl and Twist (Twi) enhances the activation of Dl target genes, while an interaction between Dl and Groucho (Gro) is essential for Dl-mediated repression. A yeast two-hybrid screen to identify Dl interacting proteins yielded, in addition to the well characterized Dl-interactors Twi and Cactus, four novel Dl-interactors (Dip1, Dip2, Dip3, and Dip4/Ubc9). Conjugation of SUMO to Dl by Ubc9 was subsequently shown to result in more potent activation by Dl (Ratnaparkhi, 2009).

Dip3 belongs to a family of proteins that contain both MADF (for Myb/SANT-like in ADF) and BESS (for BEAF, Stonewall, SuVar(3)7-like) domains. While MADF-BESS domain proteins are found in both insects and vertebrates, only a few have been characterized and their functions are largely unknown. The Drosophila genome encodes 14 MADF-BESS domain factors. In addition to Dip3, these include Adf-1, which was initially found as an activator of Alcohol dehydrogenase, and Stonewall, which is required for oogenesis. The Dip3 MADF domain mediates sequence specific binding to DNA, while the Dip3 BESS domain mediates binding to a subset of TATA binding protein associated factors as well as to the Dl RHD and to Twi. In addition to functioning as an activator, Dip3 can function as a coactivator to stimulate synergistic activation by Dl and Twi in S2 cells (Ratnaparkhi, 2009).

This study shows that Dip3 assists RHD proteins during both embryonic development and the innate immune response. By stimulating the expression of antimicrobial defense genes, Dip3 improves survival of both larvae and adults following septic injury. The presence of Dip3 near the promoters of antimicrobial defense genes depends upon Rel family proteins suggesting that Dip3 functions as a coactivator at these promoters (Ratnaparkhi, 2009).

It has been shown that Dip3, which binds both Dl and Twi via its BESS domain, synergistically enhances the activation of a luciferase reporter with multiple Dl and Twi binding sites upstream of the promoter. In addition, Dip3 has been implicated as the 'mystery protein' which binds to sites adjacent to Dl and Twi binding sites in a subset of Dl target genes. Therefore the ability of Dip3 to enhance the expression of the Dl target promoters twi, sna, and rho in S2 cell transient transfection assays was examined. All three promoters require both Dl and Twi for full activity. Dip3 was found to synergize with Dl and Twi in the activation of the sna and twi promoters, but not in the activation of the rho promoter (Ratnaparkhi, 2009).

A polyclonal antibody against recombinant Dip3 was generated, and used to determine where and when Dip3 is present in the embryo. Maternally expressed Dip3 is observed in all nuclei as early as nuclear cycle 7. It was detected in subsequent nuclear cycles during formation of the Dl nuclear concentration gradient. In interphase embryonic as well as S2 cell nuclei, Dip3 localizes to nuclear speckles of unknown identity. During mitosis Dip3 is enriched on chromosomes. It associates with the centrosome proximal portion of the anaphase chromatids and the inside ring of the polar body rosette suggesting a predominant pericentromeric location at this stage of the cell cycle and hinting at a possible role of Dip3 in centromeric function. Confirming the specificity of the antibodies, the immunoreactivity is absent from Dip31 embryos in which the Dip3 transcriptional and translational start sites as well as a large segment of the Dip3 coding region have been deleted. Weak Dip3 expression is also detected in the fat body (Ratnaparkhi, 2009).

Homozygous Dip31 flies are viable and fertile, indicating that Dip3 cannot have an essential role in embryonic D/V pattern formation. However, a small proportion (7±4%) of the embryos fail to hatch and exhibit D/V patterning defects. Embryos produced by females transheterozygous for Dip31 and a deficiency that removes a portion of the second chromosome containing the Dip3 gene (Df(PC4) exhibit similar embryonic lethality (10%) and D/V patterning defects. Also, maternal overexpression of Dip3 using the Gal4-UAS system leads to 54±9 % embryonic lethality with cuticles of the dead embryos showing both anteroposterior and D/V patterning defects, indicating that Dip3 may have a role in embryonic pattern formation (Ratnaparkhi, 2009).

Consistent with a non-essential role for Dip3 in D/V patterning, a Dip3 mutation enhances the temperature sensitive dl haploinsufficieny phenotype. The degree of dorsalization is often quantified by categorizing embryos on a scale from D0 (completely dorsalized, lacking all dorsoventral pattern elements other than dorsal epidermis) to D3 (inviable, but with little or no apparent defect in the cuticular pattern). At 29°, about half the dead embryos produced by dl1/+ females exhibit detectable D/V patterning defects and the majority of these fall into the D2 category (moderately dorsalized, exhibiting mildly expanded ventral denticle belts and a twisted germ band). Removal of maternal Dip3 increases the proportion of dorsalized embryos to about 75% with most of the increase being due to an increase in the number of D2 embryos. The effect seems to be strictly maternal as the paternal genotype does not modulate the dl haploinsufficiency phenotype (Ratnaparkhi, 2009).

Dip3 is present in the fat body, the organ in which RHD factors activate antimicrobial defense genes in response to infection. Since Dip3 binds the Dl RHD, the role of Dip3 in the innate immune response was examined by assessing the sensitivity of Dip31 flies to bacterial and fungal infection. Wild-type and Dip31 adults and larvae were injected with gram positive bacteria (M. luteus), gram negative bacteria (E. coli), and fungi (B. brassiana). For comparison, flies were infected that contained mutations in known components of the Toll (spzrm7) and Imd (RelE20) pathways. Wild-type, RelE20, spzrm7, and Dip31 adults showed little lethality (<15%) 30 days after mock infection. However, the Dip31 adult flies exhibited 55% lethality one month after injection with a 1:1 mixture of M. luteus and E. coli, compared to 10% lethality after 30 days for wild-type flies and 98% after 30 days for RelE20 flies. In contrast, wild-type and Dip31 adults were equally sensitive to fungal infection, both showing 55-70% lethality after 30 days compared to 100% lethality after 22 days for RelE20 adults and 100% lethality after 7 days for spzrm7 adults. Similar results were seen in larvae in which Dip31, RelE20 and spzrm7 mutations resulted in reduced rates of eclosion following septic injury compared to wild-type. The effectiveness of the immune challenge was further verified by an experiment showing that septic injury leads to translocation of Dl into the nucleus (Ratnaparkhi, 2009).

To determine if the sensitivity of Dip31 flies to infection results from reduced induction of antimicrobial peptides, the expression of dipt, drs and cec-A was monitored as a function of time following septic injury. Relative to uninfected flies, the levels of expression of drs and dipt were reduced by the Dip31 mutation, especially at the 2 and 4 hr time points, while the levels of cec-A expression were not significantly altered. Thus, some, but not all, antimicrobial defense genes that are regulated by RHD family proteins exhibit dependence on Dip3. At the 4 hr time point, relative to infected, wild type flies, the spzrm7 mutation reduced drs expression to basal levels while the RelE20 mutation reduced dipt expression ten fold (Ratnaparkhi, 2009).

Dip3 was over expressed in the larvae using the Cg-Gal4 driver to examine the effect of increasing levels of Dip3 on the expression of antimicrobial defense genes in the fat body. Cec-A and drs levels were unaffected, while dipt levels increased two-fold in infected flies. Thus, both loss-of-function and over expression data are consistent with the conclusion that Dip3 makes the immune response more robust by elevating the expression of a subset of antimicrobial defense genes (Ratnaparkhi, 2009).

Radiolabeled Dip3 interacts with FLAG-tagged Dl and Rel immobilized on anti-FLAG beads. Similarly, immobilized FLAG-Dip3 binds Dl (Bhaskar, 2002) and Rel (Residues 1-600). Dip3 binds to DNA via its MADF domain and to the RHD via its BESS domain, and can thus function either as an activator or as a coactivator (Bhaskar, 2002). To determine if Dip3 is present at the promoters of antimicrobial defense genes, ChIP assays were carried out in S2 cells transfected with FLAG-Dip3. FLAG antibody was used to immunoprecipitate Dip3 crosslinked to chromatin. Compared both to mock-transfected cells and to the transcribed region of a ribosomal protein-encoding gene (rp49), Dip3 was highly enriched at the drs, dipt and cecA promoters. As expected, dsRNA directed against Dip3 eliminated the ChIP signal verifying antibody specificity. The association of Dip3 with the promoters of the anti-microbial defense genes depended on Rel family proteins, since knockdown of these proteins by dsRNAi significantly reduced association of Dip3 with the promoters. Similar results were observed with an anti-GFP antibody and cells expressing a Dip3-GFP fusion protein (Ratnaparkhi, 2009).

These results suggest that Dip3 may synergize with RHD proteins in multiple developmental contexts possibly through contact with the Dl rel homology domain. Dip3 is expressed maternally and present in cleavage stage nuclei at the time that Dl is functioning to pattern the D/V axis. Furthermore, Dip3 can potentiate Dl-mediated activation of the twist and snail promoters in S2 cells. These observations suggest that Dip3 might have a role in D/V patterning. Consistent with this possibility, it was found that removal of maternal Dip3 results in occasional D/V patterning defects and significantly enhances the dl haploinsufficiency phenotype suggesting the Dip3 renders D/V patterning more robust perhaps by assisting in Dl-mediated activation (Ratnaparkhi, 2009).

An important aspect of the immune response is activation in the fat body of genes encoding antimicrobial peptides by the Rel family transcription factors Dl, Dif, and Rel. This study found that synergistic killing of flies by a mixture of E.coli and M. luteus is enhanced in Dip31 flies. This suggests roles for Dip3 in the Imd and/or Toll pathways, which mediate the response to microbial infection. In accord with this idea, it was found that activation of the Imd pathway target dipt and the Toll pathway target drs are compromised in Dip3 mutant larvae (Ratnaparkhi, 2009).

To determine if the role of Dip3 at antimicrobial defense gene promoters is direct, ChIP assays were carried out demonstrating that this factor associates directly with the drs, dipt, and cec-A promoters in S2 cells. Since Dip3 contains a DNA binding domain, it is possible that it binds to these promoters through a direct interaction with DNA. However, with one exception in the drs promoter, these promoters lack matches for the consensus Dip3 binding sites. Thus, Dip3 may be acting as a coactivator at these promoters consistent with its ability to bind the rel homology domain. In support of this idea, it was found that simultaneous knockdown of all three rel family proteins significantly reduced recruitment of Dip3 to the promoters (Ratnaparkhi, 2009).

The mechanism of Dip3 co-activation remains unclear. The finding that the Dip3 BESS domain binds TAFs (Bhaskar, 2002) suggests a role for Dip3 in the recruitment of the basal machinery. In addition, the MADF domain is closely related to the SANT domain, which binds histone tails and may have a role in interpreting the histone code. While analysis of RHD targets suggests roles for Dip3 in activation, Dip3 also associates with pericentromeric heterochromatin during mitosis, consistent with a possible role in silencing. Other heterochromatic proteins including a suppressor of position effect variegation (Su(Var)3-7) also contain BESS domains. However, the loss of Dip3 does not appear to modify position effect variegation (Ratnaparkhi, 2009).

In flies, additional roles for RHD-mediated activation have been demonstrated in haematopoesis, neural fate specification, and glutamate receptor expression. Antimicrobial defense genes are also expressed constitutively in barrier epithelia and in the male and female reproductive tracts. It will be interesting to determine if Dip3 is involved in rel protein-dependent and independent gene activation in some or all of these tissues. One tissue in which Dip3 appears to have clear rel-independent functions is in the developing compound eye, where Dip3 overexpression results in conversion of eye to antenna, while Dip3 loss-of-function leads to mispatterning of the retina (Ratnaparkhi, 2009 and references therein).

Core promoter functions in the regulation of gene expression of Drosophila dorsal target genes

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g., the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. This study explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. Over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. Multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, The activation of key Dorsal target genes by Dorsal was analyzed, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, this study demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, these results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes (Zehavi, 2014a).

This study demonstrates that the DPE is an important, conserved transcription element shared by multiple Dorsal target genes, which comprise the dorsal-ventral gene regulatory network. Specifically, over two-thirds of the known Dorsal target genes contain DPE motifs, which is significantly higher than the percentage of DPE promoters in Drosophila genes. Remarkably, only less than 8% of the Dorsal target genes contain TATA box elements without DPE motifs. The DPE is most prevalent in mesodermal Dorsal targets. The number of DPE containing genes decreases in the neuroectoderm and further decreases in the dorsal ectoderm, where the Dorsal nuclear concentration is lowest. On the other hand, the number of genes containing a TATA box is higher in regions where Dorsal nuclear concentration is decreased. The occurrence of the DPE in many developmentally regulated genes as well as analysis of the frequencies of core promoter elements in Drosophila genes and the identification of DPE motifs in the majority of Dorsal targets, imply that the DPE is not randomly distributed in ~23% of the genes; rather it is enriched in specific GRNs and pathways (Zehavi, 2014a).

This study has examined the transcription of the natural enhancers and promoters of twi, lea, tin, and brk in Drosophila Schneider S2R+ cells and have discovered that the basal transcription levels of twi, lea, tin, and brk in the absence of ectopically expressed Dorsal are highly dependent on the DPE motif (Zehavi, 2014a).

The DPE core promoter motif is an important regulatory component in Dorsal target genes in S2R+ cells. The brk and twi core promoters are dependent on the DPE motif and could not be fully activated in S2R+ cells by an added TATA box, whereas the lea core promoter is functionally dependent on the DPE, but its activity could be restored via an added TATA box (Zehavi, 2014a).

Unlike the results obtained using Drosophila S2R+ cells, in vitro transcription analysis using nuclear extracts derived from Drosophila embryos has demonstrated that the DPE motif, which is important for the transcriptional activity of twi, lea, and brk, can be replaced by a TATA box. This demonstrates the strength of the TATA box, which can restore transcription of some mDPE-containing promoters. Nevertheless, the TATA box is only naturally used in a minority of Dorsal target genes (Zehavi, 2014a).

Importantly, the mDPE and mDPE + TATA reporter constructs of twi, brk, and lea have comparable strength in vitro, yet they do not have the same strength in S2R+ cells. This suggests that there are additional DPE specificity factors in the cells. The identification of such DPE specificity factor(s) that act in the activation of twi and brk, but not in lea, awaits future investigation. Taken together, these data indicate that the core promoter contributes to the overall transcription levels and adds an important regulatory dimension to the complex dorsal-ventral gene network (Zehavi, 2014a).

This study has demonstrated that multiple Dorsal target genes are dependent on the DPE and that Dorsal has the ability to preferentially activate some of its targets via the DPE. It is interesting to note that Relish, another Rel family transcription factor, which is important for the activation of the Imd (Immune deficiency) pathway of innate immunity and binds the same or nearly identical DNA sequence motifs as Dorsal, activates transcription in a similar manner to Dorsal, albeit to a lower extent, presumably because of the lack of additional factors that might be missing in the S2R+ cells in which the Imd pathway has not been activated (Zehavi, 2014a).

In a separate study, it was observed that the maternal sequence-specific transcription factor Bicoid activates its natural target gene giant, which contains functional TATA box, Inr, and DPE motifs, regardless of its core promoter composition. The current study shows that Bicoid activates the mDPE brk reporter, highlighting the fact that the ability of Dorsal, Relish, and Caudal to activate transcription of some of their target genes with a preference for the core promoter composition is not a general property of sequence-specific transcription factors; rather, it is a unique feature of these specific transcription factors (Zehavi, 2014a).

The present study demonstrates that the DPE is broadly used in the regulation of genes that mediate the formation of the dorsal-ventral axis during early embryonic development. Furthermore, the findings highlight the importance of the core promoter, in addition to sequence-specific DNA binding motifs in enhancers, in the regulation of gene expression. It was previously demonstrated that the core promoters of the majority of the Hox genes, which are key regulators of the development of the embryonic body plan, contain functional DPE motifs. It was also shown that Caudal, a key regulator of the Hox gene network and a sequence-specific enhancer binding transcription factor, activates transcription with a distinct preference for the DPE over the TATA box. It is now proposed that the prevalence of the DPE in developmentally regulated genes provides these genes with certain advantages in the complex regulation of gene expression (e.g., kinetics of responsiveness to specific signals) (Zehavi, 2014a).

The concept of a specialized transcription system has been articulated with regards to the TCT core promoter element, which has been shown to play a key role in a system that is directed toward the synthesis of ribosomal proteins. Transcription of TATA-dependent genes is different from transcription of DPE-dependent genes in many respects, e.g. the necessary basal transcription factors, the existence of core promoter-specific enhancers, and the necessity for TBP for TATA transcription as opposed to DPE transcription, where TBP exerts inhibitory effects. Taken together, DPE transcription may be regarded as a specialized transcription system that is directed toward development (Zehavi, 2014a).

Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. This study shows that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, this study defines a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP (Shields, 2022).

Since the discovery of the original Toll gene in Drosophila (Toll-1 hereafter), a large number of Toll-related genes have been identified in both insects and mammals. The Drosophila genome encodes a total of 9 Toll-related genes, including Toll-1, while mammalian genomes encode between 10 and 13 Toll-like receptors (TLRs). TLRs are single-pass transmembrane proteins that upon ligand stimulation usually trigger a conserved intracellular signaling pathway, culminating in the activation of NF-κB transcription factors. Initially identified as an essential gene for dorsoventral patterning in the early Drosophila embryo, Toll-1 was later also found to be a critical component for innate immunity. In this function, Toll-1 signaling via the NF-κB transcription factors Dorsal and Dorsal-related immunity factor (Dif) induces the expression of anti-microbial peptides (AMPs) that mediate innate immunity. A role in innate immunity has also been demonstrated for all mammalian TLRs. Likewise, Drosophila Toll-8 (aka Tollo) regulates immunity in the trachea. Toll-7 may regulate anti-viral responses, albeit through an NF-κB-independent mechanism. However, for the remaining Toll-related proteins in Drosophila, a function in innate immunity has not been clearly demonstrated (Shields, 2022).

Of particular interest is Toll-9 in Drosophila because it behaves genetically most similar to Toll-1, and its intracellular TIR domain is most closely related to those of the mammalian TLRs. Overexpression of Toll-9 results in the production of AMPs, which led to the proposal that Toll-9 might be involved in innate immunity. However, a loss-of-function analysis with a defined Toll-9 null allele did not confirm this prediction. Nevertheless, although Drosophila Toll-9 does not appear to be directly involved in regulating expression of AMPs, it has been implicated together with a few other Toll-related proteins in cell competition, an organismal surveillance program that monitors cellular fitness and eliminates cells of reduced fitness (losers). Depending on the type of cell competition, Toll-9 participates in the expression of the pro-apoptotic genes reaper and hid in loser cells, triggering their elimination . Therefore, it has been proposed that the function of Toll signaling for elimination of bacterial pathogens by AMPs and for elimination of unfit cells by pro-apoptotic genes bears a conceptual resemblance between innate immunity and cell competition (Shields, 2022).

Apoptosis is an evolutionarily well-conserved process of cellular suicide mediated by a highly specialized class of Cys-proteases, termed caspases. In Drosophila, the pro-apoptotic genes reaper and hid promote the activation of the initiator caspase Dronc (caspase-9 homolog in Drosophila). Dronc activates the effector caspases DrICE and Dcp-1 (caspase-3 and caspase-7 homologs), which trigger the death of the cell. However, caspases not only induce apoptosis but can also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), during which caspases in apoptotic cells promote the proliferation of surviving cells independently of their role in apoptosis. Early work has shown that AiP requires the initiator caspase Dronc (Shields, 2022).

To reveal the mechanism of AiP, this study expressedthe pro-apoptotic gene hid and the apoptosis inhibitor p35 simultaneously using the ey-Gal4 driver (referred to as ey > hid,p35) which drives expression in the larval eye disc anterior to the morphogenetic furrow (MF) . p35 encodes a specific inhibitor of the effector caspases DrICE and Dcp-1 but does not block the activity of Dronc. In ey > hid,p35-expressing discs, the apoptotic pathway is activated by hid expression but blocked downstream because of DrICE and Dcp-1 inhibition by P35, rendering cells in an 'undead' condition. Although apoptosis is inhibited in undead tissue, AiP still occurs because of non-apoptotic signaling by the initiator caspase Dronc, triggering hyperplasia of the anterior portion of the larval eye imaginal discs at the expense of the posterior eye field, which is reduced in size. Combined, these effects result in overgrowth of the adult head capsule, but a reduction or even absence of the eyes in the adult animal (Shields, 2022).

Using the undead model, this study showed that AiP is mediated by extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox. ROS trigger the recruitment of hemocytes, Drosophila immune cells most similar to mammalian macrophages, to undead imaginal discs. Hemocytes in turn release signals that stimulate JNK activity in undead cells, which then promotes AiP. In addition, JNK can also induce the expression of hid, thus setting up an amplification loop in undead cells which continuously signals for AiP (Shields, 2022).

Given that undead cells are abnormal cells with potentially altered cellular fitness and that signaling by Toll-related proteins surveilles cellular fitness, this study examined if signaling by Toll-9 has an important function for undead AiP. This study shows that Toll-9 is strongly up-regulated in undead cells and is necessary for the overgrowth of undead tissue. Overexpression of Toll-9 with p35 induces all hallmarks of undead AiP signaling, including Duox-dependent ROS generation, hemocyte recruitment and JNK signaling. Mechanistically, genetic evidence is provided for a heterologous interaction between Toll-9 and Toll-1, which engages the canonical Toll-1 signaling pathway to promote nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces the expression of reaper and hid. This activity contributes to the establishment of the feedback amplification loop that signals continuously for AiP. In conclusion, although Toll-9 does not appear to have an important function in innate immunity, it appears to be involved in the expression of pro-apoptotic genes such as reaper and hid in stress situations such as cell competition and undead AiP (Shields, 2022).

Toll-9 is the most closely related Drosophila TLR compared with mammalian TLRs, but a biological function of Toll-9 has not been clearly defined. All mammalian TLRs are involved in innate immunity; therefore, the close homology has led to the prediction that Drosophila Toll-9 also participates in innate immunity. However, in Toll-9 mutants, the basal as well as bacterially induced AMP production is not affected, leading to the conclusion that Toll-9 is not involved in innate immunity (Narbonne-Reveau, 2011). Nevertheless, instead of eliminating foreign pathogens, previous work has shown that Toll-9 together with several other Toll-related receptors participates in elimination of unfit cells during cell competition. This is achieved through the expression of the pro-apoptotic gene rpr. This study demonstrates that Toll-9 has a similar rpr- and hid-inducing function during AiP, thereby adding to the database of Toll-9 function (Shields, 2022).

To identify the mechanism by which Toll-9 participates in AiP, advantage was taken of the observation that misexpression of Toll-9 is sufficient to induce overgrowth of ey > p35 animals. There are multiple aspects of this phenotype that are worth being discussed. First, key for many of the observations presented in this paper is the presence of P35, a very efficient inhibitor of the effector caspases DrICE and Dcp-1. In the absence of P35, overexpression of Toll-9 does not induce any obvious phenotypes in eye discs or adult heads. The exact reason for this P35 dependence is currently unknown, but it has also been observed upon misexpression of other genes involved in AiP, such as Myo1D, Toll-1, and SpzAct. The only known function of P35 is to inhibit DrICE and Dcp-1. Therefore, one possible explanation for the P35 dependence is that these caspases cleave and inactivate an as yet unidentified component of the AiP network, possibly to block inappropriate AiP under normal conditions. In the presence of P35, the AiP-blocking activity of DrICE is inhibited and with the addition of an AiP-inducing stimulus such as misexpression of Toll-9, AiP is engaged and can trigger tissue overgrowth. Second, the data show that ectopic p35,Toll-9 co-expression triggers overgrowth through a similar mechanism as hid,p35 co-expression. This includes Dronc activation, Duox-generated ROS, hemocyte recruitment, and JNK activation. These similarities allow placing the function of Toll-9 into the AiP network (Shields, 2022).

Third, mis-expressed Toll-9 can genetically interact with Toll-1. This interaction results in nuclear translocation of Dorsal and is dependent on Myd88, Tube, and Pelle, all canonical components of the intracellular Toll-1 signaling pathway. Importantly, the nuclear translocation of Dorsal and the p35,Toll-9-induced overgrowth is also dependent on Toll-1, suggesting that the activation of the Myd88/Tube/Pelle pathway is directly triggered by Toll-1 and not by Toll-9. Mechanistically, Toll-9 may activate Toll-1 either directly through hetero-dimerization or mediated by additional factors. Future work will be necessary to identify the molecular mechanism of the Toll-9/Toll-1 interaction (Shields, 2022).

Fourth, the outcome of the Toll-9/Toll-1 interaction is the expression of the pro-apoptotic genes reaper and hid. Because Toll-9 expression is strongly up-regulated in undead cells, these data suggest that Toll-9-induced expression of reaper and hid in undead cells is setting up an amplification loop. The cause of the strong transcriptional upregulation of Toll-9 in undead cells is unknown, but it requires JNK activity. The Toll-9 amplification loop contributes to the strength of undead signaling during AiP and propels the overgrowth of the tissue (Shields, 2022).

Fifth, another important question is how Toll signaling becomes activated during AiP. Toll-1 is activated by the ligand Spatzle during embryogenesis and the immune response. Spz requires proteolytic processing for activation, which during the immune response is mediated by the Ser-protease Spatzle-processing enzyme (SPE). Consistently, SPE RNAi can suppress both the ey > p35,Toll-9INTRA and ey > hid,p35-induced overgrowth phenotypes. SPE RNAi suppressed ey > p35,Toll-9INTRA, which lacks the extracellular domain and should be insensitive to a ligand. Thus, the suppression of ey > p35,Toll-9INTRA suggests that SPE does not act through Toll-9 but instead on Toll-1. This result is consistent with a recent finding that Toll-9 RNAi cannot suppress apoptosis induced by a dominant active SPE (SPEAct) transgene. Although that there is an unknown Toll-9 ligand cannot be ruled out, Toll-9 may not need to be activated by a ligand. Toll-9 naturally carries an amino acid substitution in the cysteine-rich extracellular domain similar to the gain-of-function Toll-11 mutant. Indeed, Toll-9 behaves as a constitutively active receptor in cell culture assays. As TLRs can form homo- and heterodimers, it is possible that the constitutive activity of Toll-9 and the strong transcriptional upregulation of Toll-9 together with ligand stimulation of Toll-1 by Spz is sufficient for the activation of the Toll-1/Toll-9 complex. However, it remains unknown how SPE becomes activated during AiP (Shields, 2022).

With these considerations in mind, the following model for Toll-9 function during undead AiP emerges. The initial stimulus for AiP is the Gal4-induced expression of hid and p35, which leads to the activation of Dronc. Because of P35, Dronc cannot induce apoptosis but instead activates Duox for generation of ROS. ROS attract hemocytes which release signals for JNK activation in undead cells. JNK signaling directly or indirectly induces Toll-9 transcription. Up-regulated Toll-9 interacts with Toll-1, and after Spz ligation the Myd88/Tube/Pelle pathway triggers the nuclear accumulation of Dorsal and potentially Dif. These factors transcriptionally induce reaper and hid expression setting up the feedback amplification loop, which maintains and propels AiP and overgrowth (Shields, 2022).

One other interesting question to examine in the future will be how the intracellular pathway of Toll-1 signaling including Dorsal and Dif can induce different target genes under different conditions. For dorsoventral patterning of the Drosophila embryo, Dorsal induces the expression of twist and snail). During immune responses in the fat body, it promotes the expression of AMP genes as well as Kennedy pathway genes for the synthesis of phospholipids, while during cell competition and AiP which occur in larval imaginal discs, pro-apoptotic genes hid and rpr are induced. One potential answer to this question is that the specificity of Toll-1 signaling may be modified by the interaction with Toll-9. Although this interaction occurs at the plasma membrane, it also might influence the activity in the nucleus. It will also be interesting to examine if Toll-1 can interact with some or all of the other Toll-related receptors in Drosophila and how this interaction might influence the specificity of the transcriptional outcome. Although Toll-9 in Drosophila does not appear to be required for innate immunity, on the basis of its non-essential function to induce AMP production during bacterial infection (Narbonne-Reveau, 2011), the currenrt work and work by others (Meyer, 2014) reveals that Toll-9 may have a function during stress responses which involves expression of pro-apoptotic genes such as rpr and hid. That was demonstrated previously for cell competition and now for undead AiP, indicating potential similarities between cell competition and undead AiP. At first, such similarities appear to be at odds with the common dogmas that proliferating winner cells trigger apoptosis of loser cells, while during AiP, apoptotic cells induce proliferation of surviving cells. However, it has also been reported that loser cells have a much more active role during cell competition and can promote the winner status of cells with increased fitness. Therefore, there appear to be significant similarities between cell competition and undead AiP. The common denominator for both systems is the expression of pro-apoptotic genes. These responses have different outcomes in both systems. During cell competition, this response involves the death of the loser cells. During undead AiP, it sets up the amplification loop known to operate in undead cells, which propels hyperplasia and tissue overgrowth (Shields, 2022).

This work was performed largely under undead conditions (i.e., in the presence of the effector caspase inhibitor p35, which is not an endogenous gene in Drosophila). In reality, however, in the absence of p35, effector caspases are also activated in apoptotic cells, which will eventually lead to the death of the cell. Therefore, the question arises as to how apoptosis and AiP are linked to allow compensatory proliferation under normal conditions. Recently, evidence has been presented that certain apoptotic cells (dying enterocytes in the adult intestine) can adopt a transiently undead-like state that enables them to signal for AiP before they are dying and removed. The transiently undead-like state is achieved by transient localization of Dronc to the plasma membrane, which might serve as a non-apoptotic compartment of the cell. In that way, apoptotic cells, before they die, can trigger AiP in a p35-independent manner. Therefore, in future research, it will be important to examine if the Toll-9/Toll-1 interaction sets up a similar amplification loop in transiently undead enterocytes (Shields, 2022).


Amino Acids - 678

Structural Domains

The Dorsal protein has a large N-terminal region of 294 amino acids, homologous to the vertebrate c-rel and its corresponding viral oncogene V-rel, the transforming gene of the reticuloendotheliosis virus strain T (Steward, 1987).

An in vivo structure-function analysis of Dorsal has been performed in order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus. All these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolishes the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region (Govind, 1996).

dorsal continued: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
date revised: 24 June 2023 

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