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Computational analysis of core promoters in the Drosophila genome
Uwe Ohler, Guo-chun Liao, Heinrich Niemann, Gerald M Rubin
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-12-research0087
Abstract: We identified TSS candidates for about 2,000 Drosophila genes by aligning 5' expressed sequence tags (ESTs) from cap-trapped cDNA libraries to the genome, while applying stringent criteria concerning coverage and 5'-end distribution. Examination of the sequences flanking these TSSs revealed the presence of well-known core promoter motifs such as the TATA box, the initiator and the downstream promoter element (DPE). We also define, and assess the distribution of, several new motifs prevalent in core promoters, including what appears to be a variant DPE motif. Among the prevalent motifs is the DNA-replication-related element DRE, recently shown to be part of the recognition site for the TBP-related factor TRF2. Our TSS set was then used to retrain the computational promoter predictor McPromoter, allowing us to improve the recognition performance to over 50% sensitivity and 40% specificity. We compare these computational results to promoter prediction in vertebrates.There are relatively few recognizable binding sites for previously known general transcription factors in Drosophila core promoters. However, we identified several new motifs enriched in promoter regions. We were also able to significantly improve the performance of computational TSS prediction in Drosophila.Transcription initiation is one of the most important control points in regulating gene expression [1,2]. Recent observations have emphasized the importance of the core promoter, a region of about 100 base-pairs (bp) flanking the transcription start site (TSS), in regulating transcription [3,4]. The core promoter serves as the recognition site for the basal transcription apparatus, which comprises the multisubunit RNA polymerase II and several auxiliary factors. Core promoters show specificity both in their interactions with enhancers and with sets of general transcription factors that control distinct subsets of genes. Although there are no known DNA sequence motifs that are shared by all core promoter
Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19  [PDF]
Muriel Boube ,Bruno Hudry,Clément Immarigeon,Yannick Carrier,Sandra Bernat-Fabre,Samir Merabet,Yacine Graba,Henri-Marc Bourbon ,David L. Cribbs
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004303
Abstract: Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded “co-factor” that acts together with Hox proteins through their homeodomains in regulated developmental transcription.
Identification of Early Zygotic Genes in the Yellow Fever Mosquito Aedes aegypti and Discovery of a Motif Involved in Early Zygotic Genome Activation  [PDF]
James K. Biedler, Wanqi Hu, Hongseok Tae, Zhijian Tu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033933
Abstract: During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2–4 hr. We used transcriptome sequencing to identify the “pure” (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0–2, 2–4, 4–8, and 8–12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2–4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2–3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.
Optimized Position Weight Matrices in Prediction of Novel Putative Binding Sites for Transcription Factors in the Drosophila melanogaster Genome  [PDF]
Vyacheslav Y. Morozov, Ilya P. Ioshikhes
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0068712
Abstract: Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. DNA-binding proteins often show degeneracy in their binding requirement and thus the overall binding specificity of many proteins is unknown and remains an active area of research. Although existing PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. Our previous study introduced a promising approach to PWM refinement in which known motifs are used to computationally mine putative binding sites directly from aligned promoter regions using composition of similar sites. In the present study, we extended this technique originally tested on single examples of transcription factors (TFs) and showed its capability to optimize PWM performance to predict new binding sites in the fruit fly genome. We propose refined PWMs in mono- and dinucleotide versions similarly computed for a large variety of transcription factors of Drosophila melanogaster. Along with the addition of many auxiliary sites the optimization includes variation of the PWM motif length, the binding sites location on the promoters and the PWM score threshold. To assess the predictive performance of the refined PWMs we compared them to conventional TRANSFAC and JASPAR sources. The results have been verified using performed tests and literature review. Overall, the refined PWMs containing putative sites derived from real promoter content processed using optimized parameters had better general accuracy than conventional PWMs.
The Selectivity and Specificity of Autophagy in Drosophila  [PDF]
Ioannis P. Nezis
Cells , 2012, DOI: 10.3390/cells1030248
Abstract: Autophagy is a process of cellular self-degradation and is a major pathway for elimination of cytoplasmic material by the lysosomes. Autophagy is responsible for the degradation of damaged organelles and protein aggregates and therefore plays a significant role in cellular homeostasis. Despite the initial belief that autophagy is a nonselective bulk process, there is growing evidence during the last years that sequestration and degradation of cellular material by autophagy can be accomplished in a selective and specific manner. Given the role of autophagy and selective autophagy in several disease related processes such as tumorigenesis, neurodegeneration and infections, it is very important to dissect the molecular mechanisms of selective autophagy, in the context of the system and the organism. An excellent genetically tractable model organism to study autophagy is Drosophila, which appears to have a highly conserved autophagic machinery compared with mammals. However, the mechanisms of selective autophagy in Drosophila have been largely unexplored. The aim of this review is to summarize recent discoveries about the selectivity of autophagy in Drosophila.
A motif-independent metric for DNA sequence specificity
Luca Pinello, Giosuè Lo Bosco, Bret Hanlon, Guo-Cheng Yuan
BMC Bioinformatics , 2011, DOI: 10.1186/1471-2105-12-408
Abstract: We present a simple, unbiased quantitative measure for DNA sequence specificity called the Motif Independent Measure (MIM). By analyzing both simulated and real experimental data, we found that the MIM measure can be used to detect sequence specificity independent of presence of transcription factor (TF) binding motifs. We also found that the level of specificity associated with H3K4me1 target sequences is highly cell-type specific and highest in embryonic stem (ES) cells. We predicted H3K4me1 target sequences by using the N- score model and found that the prediction accuracy is indeed high in ES cells.The software to compute the MIM is freely available at: https://github.com/lucapinello/mim webcite.Our method provides a unified framework for quantifying DNA sequence specificity and serves as a guide for development of sequence-based prediction models.Of the entire 3GB human genome, only about 2% codes for proteins. The identification of biological functions of the entire genome remains a major challenge [1,2]. One powerful venue to gain functional insights is to identify the proteins that bind to each genomic region. Recent development of chromatin immunoprecipitation followed by microarray or sequencing (ChIP- chip or ChIPseq) technologies has made it feasible to map genome-wide protein-DNA interaction profiles [3-5]. The data generated by these experiments have not only greatly facilitated the genome-wide characterization of regulatory elements such as enhancers [6,7] but also been integrated with other data sources to build gene regulatory networks [8-11].An important question is to what extent a specific protein-DNA interaction is mediated at the level of genomic sequences. While it is well known that specific sequence motifs are crucial for transcription factors (TF) mediated cis-regulation, there are many other proteins, such as chromatin modifiers, whose target sequences cannot simply be characterized by a handful of distinct motifs [12]. Such sequences are
The EH1 motif in metazoan transcription factors
Richard R Copley
BMC Genomics , 2005, DOI: 10.1186/1471-2164-6-169
Abstract: Using motif identification methods and database searching techniques, I delimit which homeobox and forkhead domain containing proteins also have likely EH1 motifs. I show that despite low database search scores, there is a significant association of the motif with transcription factor function. I further show that likely EH1 motifs are found in combination with T-Box, Zinc Finger and Doublesex domains as well as discussing other plausible candidate associations. I identify strong candidate EH1 motifs in basal metazoan phyla.Candidate EH1 motifs exist in combination with a variety of transcription factor domains, suggesting that these proteins have repressor functions. The distribution of the EH1 motif is suggestive of convergent evolution, although in many cases, the motif has been conserved throughout bilaterian orthologs. Groucho mediated repression was established prior to the evolution of bilateria.The Engrailed Homology 1 (EH1) motif is a short (<10 amino acids) region, initially found in engrailed (en) and other homeobox containing proteins, that mediates transcriptional repression via interaction with the WD40 repeat containing groucho (Gro) [1,2]. Shimeld [3] proposed that the EH1 motif of Smith and Jaynes was shared with various forkhead (FH/HNF-3) containing transcription factors. The short size of the motif, however, suggests that it may occur by chance in many different protein families. Shimeld did not demonstrate statistically significant sequence similarity between the motifs from the homeobox- and forkhead-containing families. However, the human orthologs of groucho (the transducin-like enhancer of split proteins) have been shown to interact with FOXA2 via a region of sequence containing an EH1 motif, clearly demonstrating the biological relevance of the sequence similarity [4].In this article I search systematically for instances of the EH1 motif in homeobox and forkhead containing genes and go on to demonstrate that the EH1 motif is also found in p
Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules  [PDF]
Feng Liu,James W. Posakony
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002796
Abstract: In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities.
Contribution of Transcription Factor Binding Site Motif Variants to Condition-Specific Gene Expression Patterns in Budding Yeast  [PDF]
Joshua S. Rest, Kevin Bullaughey, Geoffrey P. Morris, Wen-Hsiung Li
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0032274
Abstract: It is now experimentally well known that variant sequences of a cis transcription factor binding site motif can contribute to differential regulation of genes. We characterize the relationship between motif variants and gene expression by analyzing expression microarray data and binding site predictions. To accomplish this, we statistically detect motif variants with effects that differ among environments. Such environmental specificity may be due to either affinity differences between variants or, more likely, differential interactions of TFs bound to these variants with cofactors, and with differential presence of cofactors across environments. We examine conservation of functional variants across four Saccharomyces species, and find that about a third of transcription factors have target genes that are differentially expressed in a condition-specific manner that is correlated with the nucleotide at variant motif positions. We find good correspondence between our results and some cases in the experimental literature (Reb1, Sum1, Mcm1, and Rap1). These results and growing consensus in the literature indicates that motif variants may often be functionally distinct, that this may be observed in genomic data, and that variants play an important role in condition-specific gene regulation.
An intuitionistic approach to scoring DNA sequences against transcription factor binding site motifs
Fernando Garcia-Alcalde, Armando Blanco, Adrian J Shepherd
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-551
Abstract: We propose SCintuit, a new scoring method for measuring sequence-motif affinity based on IFS theory. Unlike existing methods that consider dependencies between positions, SCintuit is designed to prevent overestimation of less conserved positions of TFBSs. For a given pair of bases, SCintuit is computed not only as a function of their combined probability of occurrence, but also taking into account the individual importance of each single base at its corresponding position. We used SCintuit to identify known TFBSs in DNA sequences. Our method provides excellent results when dealing with both synthetic and real data, outperforming the sensitivity and the specificity of two existing methods in all the experiments we performed.The results show that SCintuit improves the prediction quality for TFs of the existing approaches without compromising sensitivity. In addition, we show how SCintuit can be successfully applied to real research problems. In this study the reliability of the IFS theory for motif discovery tasks is proven.Cells control the abundance of proteins by means of diverse mechanisms. One such mechanism is the regulation of transcription, which is a continuous process whereby many factors combine to ensure appropriate rates of protein synthesis. Understanding such complex processes is one of the main objectives in computational biology. In its early stages, transcription is controlled, among other mechanisms, by the binding of proteins called transcription factors (TFs) to specific regions of a given chromosome called transcription factor binding sites (TFBSs). These interactions between proteins and DNA usually take place upstream from the gene, close to the transcription start site (TSS), in the so-called promoter region of the gene.One of the biggest issues in identifying TFBSs is that a single binding protein can bind to different DNA sequences. Related DNA sequences to which the same TF can bind are grouped together into a TFBS motif. The identification
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