The features of Drosophila core promoters revealed by statistical analysis

BACKGROUND: Experimental investigation of transcription is still a very labor- and time-consuming process. Only a few transcription initiation scenarios have been studied in detail. The mechanism of interaction between basal machinery and promoter, in particular core promoter elements, is not known for the majority of identified promoters. In this study, we reveal various transcription initiation mechanisms by statistical analysis of 3393 nonredundant Drosophila promoters. RESULTS: Using Drosophila-specific position-weight matrices, we identified promoters containing TATA box, Initiator, Downstream Promoter Element (DPE), and Motif Ten Element (MTE), as well as core elements discovered in Human (TFIIB Recognition Element (BRE) and Downstream Core Element (DCE)). Promoters utilizing known synergetic combinations of two core elements (TATA_Inr, Inr_MTE, Inr_DPE, and DPE_MTE) were identified. We also establish the existence of promoters with potentially novel synergetic combinations: TATA_DPE and TATA_MTE. Our analysis revealed several motifs with the features of promoter elements, including possible novel core promoter element(s). Comparison of Human and Drosophila showed consistent percentages of promoters with TATA, Inr, DPE, and synergetic combinations thereof, as well as most of the same functional and mutual positions of the core elements. No statistical evidence of MTE utilization in Human was found. Distinct nucleosome positioning in particular promoter classes was revealed. CONCLUSION: We present lists of promoters that potentially utilize the aforementioned elements/combinations. The number of these promoters is two orders of magnitude larger than the number of promoters in which transcription initiation was experimentally studied. The sequences are ready to be experimentally tested or used for further statistical analysis. The developed approach may be utilized for other species

Signal Transducers and Activators of Transcription-1 (STAT1) Regulates microRNA Transcription in Interferon γ-Stimulated HeLa Cells

Constructing and modeling the gene regulatory network is one of the central themes of systems biology. With the growing understanding of the mechanism of microRNA biogenesis and its biological function, establishing a microRNA-mediated gene regulatory network is not only desirable but also achievable.In this study, we propose a bioinformatics strategy to construct the microRNA-mediated regulatory network using genome-wide binding patterns of transcription factor(s) and RNA polymerase II (RPol II), derived using chromatin immunoprecipitation following next generation sequencing (ChIP-seq) technology. Our strategy includes three key steps, identification of transcription start sites and promoter regions of primary microRNA transcripts using RPol II binding patterns, selection of cooperating transcription factors that collaboratively function with the transcription factors targeted by ChIP-seq assay, and construction of the network that contains regulatory cascades of both transcription factors and microRNAs.Using CAMDA (Critical Assessment of Massive Data Analysis) 2009 data set that includes ChIP-seq data on RPol II and STAT1 (signal transducers and activators of transcription 1) in HeLa S3 cells in control condition and with interferon gamma stimulation, we first identified promoter regions of 83 microRNAs in HeLa cells. We then identified two potential STAT1 collaborating factors, AP-1 and C/EBP (CCAAT enhancer-binding proteins), and further established eight feedback network elements that may regulate cellular response during interferon gamma stimulation.This study offers a bioinformatics strategy to provide testable hypotheses on the mechanisms of microRNA-mediated transcriptional regulation, based upon genome-wide protein-DNA interaction data derived from ChIP-seq experiments

Microevolution of cis-Regulatory Elements: An Example from the Pair-Rule Segmentation Gene fushi tarazu in the Drosophila melanogaster Subgroup

The importance of non-coding DNAs that control transcription is ever noticeable, but the characterization and analysis of the evolution of such DNAs presents challenges not found in the analysis of coding sequences. In this study of the cis-regulatory elements of the pair rule segmentation gene fushi tarazu (ftz) I report the DNA sequences of ftz's zebra element (promoter) and a region containing the proximal enhancer from a total of 45 fly lines belonging to several populations of the species Drosophila melanogaster, D. simulans, D. sechellia, D. mauritiana, D. yakuba, D. teissieri, D. orena and D. erecta. Both elements evolve at slower rate than ftz synonymous sites, thus reflecting their functional importance. The promoter evolves more slowly than the average for ftz's coding sequence while, on average, the enhancer evolves more rapidly, suggesting more functional constraint and effective purifying selection on the former. Comparative analysis of the number and nature of base substitutions failed to detect significant evidence for positive/adaptive selection in transcription-factor-binding sites. These seem to evolve at similar rates to regions not known to bind transcription factors. Although this result reflects the evolutionary flexibility of the transcription factor binding sites, it also suggests a complex and still not completely understood nature of even the characterized cis-regulatory sequences. The latter seem to contain more functional parts than those currently identified, some of which probably transcription factor binding. This study illustrates ways in which functional assignments of sequences within cis-acting sequences can be used in the search for adaptive evolution, but also highlights difficulties in how such functional assignment and analysis can be carried out

Transcriptional regulation of yir genes in Plasmodium yoelii yoelii infected erythrocytes

In 1998, a large multi gene family was discovered in Plasmodium vivax (del Portillo et al., 1998). This multigene family was termed vir, and later studies showed that vir homologues existed in the three rodent malaria species, Plasmodium chabaudi (cir), Plasmodium berghei (bir) and Plasmodium yoelii (yir). By 5x coverage sequencing of Plasmodium yoelii 17X, 838 yir genes were predicted (Carlton et al., 2002), making this the largest known multi gene family in Plasmodium. YIR proteins are expressed at the surface of infected erythrocytes (Cunningham et al., 2005), and therefore this family is thought to be involved in antigenic variation. The aim of this thesis was to examine how P.yoelii regulates transcription of the yir family. The yir gene structure was verified experimentally, and phylogenetic analysis showed that yir genes could be divided into five supergroups consisting of yir genes with different sizes and subtelomeric localisation. In the blood stages, numerous yir genes were transcribed from all the supergroups in immunocompromised mice. However, a maximum of two yir genes were transcribed in single infected erythrocytes at the Schizont stage, which suggested strong silencing mechanisms. The transcriptional start and polyadenylation sites were identified experimentally, and it was found that both occurred at highly conserved motifs. In addition, the transcription initiation site was located close to an unusual and universally conserved triple-repeat motif, and it was found that all yir transcripts in two populations of parasites initiated downstream of this motif. Transfection experiments were performed in order to examine the role of this motif, but no solid conclusions could be drawn from these. Several alternative splicing events were detected in the yir 5'UTRs, and one of these led to exon 1 skipping of a yir gene. Through bioinformatic analysis of yir 5' intergenic regions, it was found that the UTR introns had a discrete distribution amongst the yir supergroups

Genomic and transcriptomic analyses of Microbotryum lychnidis-dioicae provide insights into the biology of a fascinating fungal phytopathogen.

This study made use of the Silene latifolia/Microbotryum lychnidis-dioicae phytopathogen system as the focal system to establish the first reference genome for Microbotryum violaceum sensu lato. In silico analysis was performed on the genome assembly to identify various characteristics of the genome. Using RNA-Sequencing technologies on the Illumina platform, we collected transcriptomic data for both in vitro and in planta life stages of the fungus, providing the most comprehensive look at the gene expression and regulation of this fungus. Due to a lack of identifiable domains on the predicted genes, gene set enrichment analysis was done in context, by including gene sets like “secreted proteins”, “small secreted proteins” and “unique proteins”, to aid discovery of the features in the different datasets. To further research into Microbotryum species in general, we developed, for the first time, a robust and repeatable Agrobacterium-mediated transformation system. Using genomic and transcriptomic data, we were able to select native promoters that drive transcription in specific conditions, making it a highly versatile and controllable system

Regulation and Function of Directly Repressed Targets of Wnt Signaling in Drosophila.

In multicellular organisms, cells communicate with each other through biochemical cascades known as signaling pathways. Being one of these pathways, Wnt signaling is repetitively used throughout animal development as well as in adult tissue regeneration and stem cell maintenance. Misregulation of Wnt signaling has been connected to several cancers, bone disease and metabolic disorders. While Wnt signaling has been studied for more than 30 years, many unanswered questions remain about how it influences cell fate and behavior. For instance, there has been considerable progress on understanding the basic mechanisms of how Wnt signaling regulates the transcription of its targets but how this pathway regulates different genes in distinct tissues/cell types is still poorly understood. My thesis work addressed the question of how Wnt signaling diversity is achieved by examining the Wnt-dependent regulation of Tiggrin (Tig), an essential Drosophila gene encoding an extracellular matrix protein. Unlike the vast majority of Wnt targets, which are activated by the pathway, Tig is directly repressed by Wnt stimulation. The first project of my thesis intended to better understand the mechanism of this repression. I discovered non-tranditional DNA motifs in a cis-regulatory module from the Tig locus that mediate Wnt-repression in the Drosophila hematopoietic system through bipartite recognition by TCF, the major transcription factor of the pathway. When swapped with the classic TCF binding sites, I found that the quality of TCF binding sites dictates the type of transcriptional output, i.e. activation or repression, likely through allosteric regulation of TCF. This work provides a paradigm for diversity in Wnt signaling and basic transcriptional regulation. The second project of my thesis describes the role of Tig in influencing hematopoiesis in Drosophila larva. I found that the Wee1 kinase, which slows G2/M progression, inhibits maturation of a major lineage of Drosophila immune cells through activation of Tig. This work elucidates connections between cell cycle regulators, the extracellular matrix and hematopoiesis.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133313/1/chenzh_1.pd

Doctor of Philosophy

dissertationRNA Polymerase (Pol) III transcribes small noncoding RNAs (e.g., tRNAs) important for translational capacity. Maf1 is a repressor of Pol III conserved from yeast to human, required for repression of Pol III in response to multiple environmental stresses, such as nutrient deprivation. Interestingly, Maf1 is a phosphoprotein, being phosphorylated in good growth conditions and dephosphorylated in poor growing conditions. The phosphatase acting on Maf1 has not been determined. I investigated the identity of the phosphatase in yeast Saccharomyces cerevisiae using a genetic Maf1-Pol III fusion construct in combination with molecular and biochemical assays. I queried members of Protein Phosphatase 2A and 4 complexes (PP2A and PP4, respectively) for their role in dephosphorylation of Maf1 and determined that PP4-containing Pph3 and Psy2, together with accessory factors Rrd1 and Tip41-is the major Maf1 phosphatase, acting in response to multiple stresses. Maf1 interacts with Pph3 in vivo, and biochemical purification of TAP-tagged Pph3-bound complex shows activity on purified, endogenously phosphorylated Maf1 in vitro, suggesting that PP4 is a direct phosphatase of Maf1. In human cells, regulation of Pol III also involves chromatin regulation, not apparent in studies of Pol III in yeast. The full repertoire of Pol III-transcribed genes in human cells has not been defined. I determined the full Pol III ! ! ! "#! transcriptome in human (HeLa) cells by chromatin immunoprecipitation (ChIP), coupled with microarray (ChIP-array) or high throughput sequencing (ChIP-seq), for Pol III subunit Rpc32, and Pol III transcription factors Brf1, Brf2 and TFIIIC63. I also determined the Pol III transcriptome in other cell types to address the possibility of cell type-specific activation of Pol III genes. Although many active Pol III genes were shared between all cell types assayed, a large number of genes were differentially enriched with Pol III. I compared enrichment of Pol III with chromatin modifications, transcription factors, and Pol II ChIP-seq profiles and found a significant correlation for active Pol III genes with active chromatin modifications and occupancy of transcription factors and Pol II. These results suggest that active chromatin gates Pol III accessibility to the human genome