Abstract:I for activation and (ii) Dl-AP1-B P for repression. These two subcircuits are temporally delineated and bring about overall regulation of dorsal during immune response. These results suggest the presence of a previously unknown mechanism of Dorsal autoregulation in immune-challenged Drosophila.
“…However, the major groove geometry of A 6– containing κB motifs (Figure 5B and C) is distinct from that of the T 6 motifs (Figure 5D–F). We found that these two motifs have constrained major grooves and form repressor type DNA-Dl complexes (Figure 5B, C and G, lanes 6 and 7) (39). …”
Section: Resultsmentioning
confidence: 97%
“…While dlκB I is a typical κB -motif ( GGGAATTCC ) and acts as an enhancer, atypical dlκB P ( AGAAAAACA ) acts as a repressor (39). For the functional assay, these motifs were placed at the dlκB P position in the dorsal promoter construct with a luciferase reporter (39). Figure 2.Enhancer or repressor activity of κB -motifs is encoded in its sequence.…”
Section: Resultsmentioning
confidence: 99%
“…It has been shown that a single base change in the κB -motif can determine the co-factor specificity of NFκB dimers thus highlighting the role of κB sequence in Rel-mediated gene regulation ( 38 ). Recently, we demonstrated ‘sequence specificity’ in Dl interaction with its co-regulator AP1, as we found that the Dl–AP1 complex was recruited on an atypical AGAAAAACA motif but not on canonical GGGAATTCC motif in the same promoter ( 39 ). This raised the question as to how Dl-binding motif sequence decides co-regulator specificity.…”
Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network.
“…However, the major groove geometry of A 6– containing κB motifs (Figure 5B and C) is distinct from that of the T 6 motifs (Figure 5D–F). We found that these two motifs have constrained major grooves and form repressor type DNA-Dl complexes (Figure 5B, C and G, lanes 6 and 7) (39). …”
Section: Resultsmentioning
confidence: 97%
“…While dlκB I is a typical κB -motif ( GGGAATTCC ) and acts as an enhancer, atypical dlκB P ( AGAAAAACA ) acts as a repressor (39). For the functional assay, these motifs were placed at the dlκB P position in the dorsal promoter construct with a luciferase reporter (39). Figure 2.Enhancer or repressor activity of κB -motifs is encoded in its sequence.…”
Section: Resultsmentioning
confidence: 99%
“…It has been shown that a single base change in the κB -motif can determine the co-factor specificity of NFκB dimers thus highlighting the role of κB sequence in Rel-mediated gene regulation ( 38 ). Recently, we demonstrated ‘sequence specificity’ in Dl interaction with its co-regulator AP1, as we found that the Dl–AP1 complex was recruited on an atypical AGAAAAACA motif but not on canonical GGGAATTCC motif in the same promoter ( 39 ). This raised the question as to how Dl-binding motif sequence decides co-regulator specificity.…”
Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network.
“…Recent studies in Drosophila have suggested that specificity in target recognition by Hox proteins is determined by minor groove shape while that of the Dorsal target genes is ascertained by the major groove geometry ( 3 , 28 , 29 ). This has brought focus on the shape readout of the DNA motif in determining specificity in its interaction with TFs ( 30 , 31 ).…”
Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL:
http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm
“…Targeting simultaneously several viral genes from a single transgene construct is possible since only a few hundredbasepair long double-strand RNA sequence is sufficient to elicit RNA interference in the silkworm (Gandhe et al 2007;Mrinal and Nagaraju 2010;Shukla and Nagaraju 2010;Terenius et al 2011). With the aim of installing a strong antiviral trait, we assayed the properties of a transgene carrying four tandem sequences arising from four essential genes of the baculovirus (ie1, lef1, lef3, and p74).…”
Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for .50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (.75% survival rate as compared to ,15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture. R NA interference (RNAi) is a mechanism by which cells silence the expression of foreign genes. This process often provides an adaptive innate immunity against viruses where double-stranded RNAs encoded by the viruses during infection act as pathogen trigger after they are taken up by the cellular RNAi machinery (Wang et al. 2006). Alternatively, this natural defense mechanism is exploited as an antiviral therapy via the artificial inhibition of the expression of essential viral genes (Leonard and Schaffer 2006). Multiple protocols of delivery of dsRNA or of constructs encoding dsRNAs in the organism are currently under assay to combat infections of various viruses. The efficiency of the assays is still challenged by the delicate setting of the proper dosage of the RNAi, the relative longevity of the effect, the occurrence of RNAi driven toxicity, and the virus-intrinsic susceptibility. A few attempts have been made in animal and plant models where the antiviral trait was installed by transgenesis to confer stable protection to the transformed individuals and to their progeny. Successes have been reached in plants ( Bucher et al. 2006;Bonfim et al. 2007;Zhang 2010) but, to our knowledge, no case has yet been reported in animals showing a stable and robust protection against a virus after a RNAi-aided antiviral trait was introduced through germline transformation.The baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), is a major pathogen that affects silkworm rearings and hampers silk cocoon production in Asia. In India alone .50% of silk cocoon crop losses are attributed to baculovirus infection (Khurad et al. 2006). Effective treatment against the virus has been elusive due to its sturdy nature and the lack of control strategies. Interestingly, the biology of the virus is reasonably...
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