Transcriptional Auto-Regulation of RUNX1 P1 Promoter

RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5\u27UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription

Conserved elements with potential to form polymorphic G-quadruplex structures in the first intron of human genes

To understand how potential for G-quadruplex formation might influence regulation of gene expression, we examined the 2 kb spanning the transcription start sites (TSS) of the 18 217 human RefSeq genes, distinguishing contributions of template and nontemplate strands. Regions both upstream and downstream of the TSS are G-rich, but the downstream region displays a clear bias toward G-richness on the nontemplate strand. Upstream of the TSS, much of the G-richness and potential for G-quadruplex formation derives from the presence of well-defined canonical regulatory motifs in duplex DNA, including CpG dinucleotides which are sites of regulatory methylation, and motifs recognized by the transcription factor SP1. This challenges the notion that quadruplex formation upstream of the TSS contributes to regulation of gene expression. Downstream of the TSS, G-richness is concentrated in the first intron, and on the nontemplate strand, where polymorphic sequence elements with potential to form G-quadruplex structures and which cannot be accounted for by known regulatory motifs are found in almost 3000 (16%) of the human RefSeq genes, and are conserved through frogs. These elements could in principle be recognized either as DNA or as RNA, providing structural targets for regulation at the level of transcription or RNA processing

Systematic identification of conserved motif modules in the human genome

<p>Abstract</p> <p>Background</p> <p>The identification of motif modules, groups of multiple motifs frequently occurring in DNA sequences, is one of the most important tasks necessary for annotating the human genome. Current approaches to identifying motif modules are often restricted to searches within promoter regions or rely on multiple genome alignments. However, the promoter regions only account for a limited number of locations where transcription factor binding sites can occur, and multiple genome alignments often cannot align binding sites with their true counterparts because of the short and degenerative nature of these transcription factor binding sites.</p> <p>Results</p> <p>To identify motif modules systematically, we developed a computational method for the entire non-coding regions around human genes that does not rely upon the use of multiple genome alignments. First, we selected orthologous DNA blocks approximately 1-kilobase in length based on discontiguous sequence similarity. Next, we scanned the conserved segments in these blocks using known motifs in the TRANSFAC database. Finally, a frequent pattern mining technique was applied to identify motif modules within these blocks. In total, with a false discovery rate cutoff of 0.05, we predicted 3,161,839 motif modules, 90.8% of which are supported by various forms of functional evidence. Compared with experimental data from 14 ChIP-seq experiments, on average, our methods predicted 69.6% of the ChIP-seq peaks with TFBSs of multiple TFs. Our findings also show that many motif modules have distance preference and order preference among the motifs, which further supports the functionality of these predictions.</p> <p>Conclusions</p> <p>Our work provides a large-scale prediction of motif modules in mammals, which will facilitate the understanding of gene regulation in a systematic way.</p

Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. © 2009 Corcoran et al

INTEGRATIVE GENOME-WIDE ANALYSIS OF ALTERNATIVE PRE-MRNA SPLICING REGULATION BY THE DROSOPHILA SR PROTEIN FAMILY

Alternative pre-mRNA splicing is a powerful mechanism that is exploited by higher eukaryotes to diversify their proteomes, and to differentially regulate the expression, function, and localization of mRNA and proteins. Pre-mRNA splicing is typically regulated by RNA-binding proteins that recognize cis-acting RNA elements, and either activate or repress splicing of adjacent exons in a temporal, and tissue specific, manner. Understanding how RNA-binding proteins control the splicing code is fundamental to understanding organismal development and disease. The SR proteins are a well-conserved class of RNA-binding proteins that have an essential role in the regulation of splice site selection, and have also been implicated as key regulators during other stages of RNA metabolism. The complexity of the RNA targets, and specificity of RNA binding location remains poorly understood for many members of the SR protein family. Here, we present a comprehensive study to elucidate how the SR proteins coordinate to regulate alternative pre-mRNA splicing (AS) in Drosophila. Genome-wide analysis of SR-dependent splicing by RNA-seq, reveals that SR proteins are required for the regulation of many types of alternative splicing events, and can act as positive or negative regulators of splice site choice depending on their binding location on the target RNA. In addition, a vast majority of regulated targets require multiple SR protein members for regulation. RNAi of multiple SR proteins simultaneously results in an additive change in the magnitude of splicing. This indicates that SR proteins co-regulate alternative splicing events in a combinatorial manner through binding specific locations on the target transcripts. Using single-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), we define the specific RNA-binding maps of the entire family of SR proteins in the transcriptome of Drosophila S2 cells. We find that SR proteins bind a distinct, but functionally diverse, class of RNAs that includes mRNAs, both constitutive and alternatively spliced, as well as non-coding RNAs. Closer analysis of the bound transcripts revealed that while individual SR proteins can bind unique transcripts, multiple SR protein family members bind a majority of the target transcripts. This comprehensive analysis reveals position-dependent RNA splicing maps, in vivo consensus binding motifs, and a high level of cross- and coordinated regulation of alternative splicing by the SR protein family

Expression-Guided In Silico Evaluation of Candidate Cis Regulatory Codes for Drosophila Muscle Founder Cells

While combinatorial models of transcriptional regulation can be inferred for metazoan systems from a priori biological knowledge, validation requires extensive and time-consuming experimental work. Thus, there is a need for computational methods that can evaluate hypothesized cis regulatory codes before the difficult task of experimental verification is undertaken. We have developed a novel computational framework (termed “CodeFinder”) that integrates transcription factor binding site and gene expression information to evaluate whether a hypothesized transcriptional regulatory model (TRM; i.e., a set of co-regulating transcription factors) is likely to target a given set of co-expressed genes. Our basic approach is to simultaneously predict cis regulatory modules (CRMs) associated with a given gene set and quantify the enrichment for combinatorial subsets of transcription factor binding site motifs comprising the hypothesized TRM within these predicted CRMs. As a model system, we have examined a TRM experimentally demonstrated to drive the expression of two genes in a sub-population of cells in the developing Drosophila mesoderm, the somatic muscle founder cells. This TRM was previously hypothesized to be a general mode of regulation for genes expressed in this cell population. In contrast, the present analyses suggest that a modified form of this cis regulatory code applies to only a subset of founder cell genes, those whose gene expression responds to specific genetic perturbations in a similar manner to the gene on which the original model was based. We have confirmed this hypothesis by experimentally discovering six (out of 12 tested) new CRMs driving expression in the embryonic mesoderm, four of which drive expression in founder cells

Diverse RNA-Binding Proteins Interact with Functionally Related Sets of RNAs, Suggesting an Extensive Regulatory System

RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3′-untranslated regions, others in 5′-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate

Multigenome DNA sequence conservation identifies Hox cis-regulatory elements

To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced ∼0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide

Discovering Conserved cis-Regulatory Elements That Regulate Expression in Caenorhabditis elegans

The aim of this dissertation is two-fold:: 1) To catalog all cis-regulatory elements within the intergenic and intronic regions surrounding every gene in C.elegans: i.e. the regulome) and: 2) to determine which cis-regulatory elements are associated with expression under specific conditions. We initially use PhyloNet to predict conserved motifs with instances in about half of the protein-coding genes. This initial first step was valuable as it recovered some known elements and cis-regulatory modules. Yet the results had a lot of redundant motifs and sites, and the approach was not efficiently scalable to the entire regulome of C. elegans or other higher-order eukaryotes. Magma: Multiple Aligner of Genomic Multiple Alignments) overcomes these shortcomings by using efficient clustering and memory management algorithms. Additionally, it implements a fast greedy set-cover solution to significantly reduce redundant motifs. These differences make Magma ~70 times faster than PhyloNet and Magma-based predictions occur near ~99% of all C. elegans protein-coding genes. Furthermore, we show tractable scaling for higher-order eukaryotes with larger regulomes. Finally, we demonstrate that a Magma-predicted motif, which represents the binding specificity for HLH-30, plays a critical role in the host-defense to pathogenic infections. This novel finding shows that hlh-30(-) animals are more susceptible to S. aureus and P. aeruginosa than their wild type counterparts

In silico discovery of transcription regulatory elements in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>With the sequence of the <it>Plasmodium falciparum </it>genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the <it>P. falciparum </it>genome sequence having revealed few <it>cis</it>-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of <it>P. falciparum </it>intergenic regions (~90% AT) presents significant challenges to <it>in silico cis</it>-regulatory element discovery.</p> <p>Results</p> <p>We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich <it>P. falciparum </it>genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from <it>P. falciparum </it>life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative <it>cis</it>-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the <it>Plasmodium </it>literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.</p> <p>Conclusion</p> <p>This GEMS analysis demonstrates that <it>in silico </it>regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of <it>P. falciparum</it>. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that <it>cis</it>-regulatory elements play a role in the transcriptional control of many <it>P. falciparum </it>biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.</p