Aditi Kanhere

A novel method for prokaryotic promoter prediction based on DNA stability

BackgroundIn the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. This work presents a novel prokaryotic promoter prediction method based on DNA stability.ResultsThe promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of promoter prediction based on the differences in the stability of DNA sequences in the promoter and non-promoter region works much better compared to existing prokaryotic promoter prediction programs, which are based on sequence motif searches. At present the method works optimally for genomes such as that of Escherichia coli, which have near 50 % G+C composition and also performs satisfactorily in case of other prokaryotic promoters.ConclusionsOur analysis clearly shows that the change in stability of DNA seems to provide a much better clue than usual sequence motifs, such as Pribnow box and -35 sequence, for differentiating promoter region from non-promoter regions. To a certain extent, it is more general and is likely to be applicable across organisms. Hence incorporation of such features in addition to the signature motifs can greatly improve the presently available promoter prediction programs.

Predicting transcription factor affinities to DNA from a biophysical model

MOTIVATION Theoretical efforts to understand the regulation of gene expression are traditionally centered around the identification of transcription factor binding sites at specific DNA positions. More recently these efforts have been supplemented by experimental data for relative binding affinities of proteins to longer intergenic sequences. The question arises to what extent these two approaches converge. In this paper, we adopt a physical binding model to predict the relative binding affinity of a transcription factor for a given sequence. RESULTS We find that a significant fraction of genome-wide binding data in yeast can be accounted for by simple count matrices and a physical model with only two parameters. We demonstrate that our approach is both conceptually and practically more powerful than traditional methods, which require selection of a cutoff. Our analysis yields biologically meaningful parameters, suitable for predicting relative binding affinities in the absence of experimental binding data. AVAILABILITY The C source code for our TRAP program is freely available for non-commercial use at http://www.molgen.mpg.de/~manke/papers/TFaffinities/

T-bet and GATA3 orchestrate Th1 and Th2 differentiation through lineage-specific targeting of distal regulatory elements

T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions.

Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes

During the process of transcription, RNA polymerase can exactly locate a promoter sequence in the complex maze of a genome. Several experimental studies and computational analyses have shown that the promoter sequences apparently possess some special properties, such as unusual DNA structures and low stability, which make them distinct from the rest of the genome. But most of these studies have been carried out on a particular set of promoter sequences or on promoter sequences from similar organisms. To examine whether the promoters from a wide variety of organisms share these special properties, we have carried out an analysis of sets of promoters from bacteria, vertebrates and plants. These promoters were analyzed with respect to the prediction of three different properties, such as DNA curvature, bendability and stability, which are relevant to transcription. All the promoter sequences are predicted to share certain features, such as stability and bendability profiles, but there are significant differences in DNA curvature profiles and nucleotide composition between the different organisms. These similarities and differences are correlated with some of the known facts about transcription process in the promoters from the three groups of organisms.

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

Horizontal Gene Transfers in prokaryotes show differential preferences for metabolic and translational genes

BackgroundHorizontal gene transfer (HGT) is an important process, which contributes in bacterial pathogenesis and drug resistance. A number of methods have been proposed for detection of horizontal gene transfer. One successful approach to the detection of HGT events is due to Novichkov et al. (J. Bacteriology 186, 6575–85), who rely on comparing phylogenetic distances within a gene family with genomic distances of the source organisms. Building on their approach, we introduce outlier detection in the correlation between those two sets of distances. This approach is designed to detect horizontal transfers of core set of genes present in many bacteria. The principle behind method allows detection of xenologous gene displacements as well as acquisition of novel genes.ResultsSimulations indicated that our method performs better than Novichkov et als original approach. The approach very efficiently identified HGT between distantly related bacteria and also a limited number of gene transfers between closely related bacteria. In combination with sequence similarity and likelihood tests, it yields a measure robust enough to derive a set of 171 genes deemed likely to have been horizontally transferred. Further analysis of these 171 established horizontal transfer events gave interesting insights in the direction of transfer.ConclusionThe majority of transfers between archaea and bacteria have occurred in the direction from bacteria to archaea rather than the other way round. Genes transferred between the archaea and bacteria are mostly metabolic genes. On the other hand, genes transferred within the bacterial phyla are mainly involved in translation.

CpG-depleted promoters harbor tissue-specific transcription factor binding signals—implications for motif overrepresentation analyses

Motif overrepresentation analysis of proximal promoters is a common approach to characterize the regulatory properties of co-expressed sets of genes. Here we show that these approaches perform well on mammalian CpG-depleted promoter sets that regulate expression in terminally differentiated tissues such as liver and heart. In contrast, CpG-rich promoters show very little overrepresentation signal, even when associated with genes that display highly constrained spatiotemporal expression. For instance, while ∼50% of heart specific genes possess CpG-rich promoters we find that the frequently observed enrichment of MEF2-binding sites upstream of heart-specific genes is solely due to contributions from CpG-depleted promoters. Similar results are obtained for all sets of tissue-specific genes indicating that CpG-rich and CpG-depleted promoters differ fundamentally in their distribution of regulatory inputs around the transcription start site. In order not to dilute the respective transcription factor binding signals, the two promoter types should thus be treated as separate sets in any motif overrepresentation analysis.

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

ABSTRACT Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral- and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.

Genome-Wide Regulatory Analysis Reveals That T-bet Controls Th17 Lineage Differentiation through Direct Suppression of IRF4

The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates Th cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor IFN regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet–deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the roles of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interplay of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells.

Genome-Wide Analyses of Zta Binding to the Epstein-Barr Virus Genome Reveals Interactions in both Early and Late Lytic Cycles and an Epigenetic Switch Leading to an Altered Binding Profile

ABSTRACT The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.