[show abstract][hide abstract] ABSTRACT: Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these 'long-range' enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3(rd) chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional-that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.
PLoS ONE 01/2013; 8(4):e60137. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the developing CNS, unique functional identities among neurons and glia are, in part, established as a result of successive transitions in gene expression programs within neural precursor cells. One of the temporal-identity windows within Drosophila CNS neural precursor cells or neuroblasts (NBs) is marked by the expression of a zinc-finger transcription factor (TF) gene, castor (cas). Our analysis of cis-regulatory DNA within a cas loss-of-function rescue fragment has identified seven enhancers that independently activate reporter transgene expression in specific sub-patterns of the wild-type embryonic cas gene expression domain. Most of these enhancers also regulate different aspects of cas expression within the larval and adult CNS. Phylogenetic footprinting reveals that each enhancer is made up of clusters of highly conserved DNA sequence blocks that are flanked by less-conserved inter-cluster spacer sequences. Comparative analysis of the conserved DNA also reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. Intra-species alignments show that two of the sub-pattern enhancers originated from an inverted duplication and that this repeat is unique to the cas locus in all sequenced Drosophila species. Finally we show that three of the enhancers differentially require cas function for their wild-type regulatory behavior. Cas limits the expression of one enhancer while two others require cas function for full expression. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it.
[show abstract][hide abstract] ABSTRACT: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs.
We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization.
cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.
[show abstract][hide abstract] ABSTRACT: Many of the key regulators of Drosophila CNS neural identity are expressed in defined temporal orders during neuroblast (NB) lineage development. To begin to understand the structural and functional complexity of enhancers that regulate ordered NB gene expression programs, we have undertaken the mutational analysis of the temporally restricted nerfin-1 NB enhancer. Our previous studies have localized the enhancer to a region just proximal to the nerfin-1 transcription start site. Analysis of this enhancer, using the phylogenetic footprint program EvoPrinter, reveals the presence of multiple sequence blocks that are conserved among drosophilids. cis-Decoder alignments of these conserved sequence blocks (CSBs) has identified shorter elements that are conserved in other Drosophila NB enhancers. Mutagenesis of the enhancer reveals that although each CSB is required for wild-type expression, neither position nor orientation of the CSBs within the enhancer is crucial for enhancer function; removal of less-conserved or non-conserved sequences flanking CSB clusters also does not significantly alter enhancer activity. While all three conserved E-box transcription factor (TF) binding sites (CAGCTG) are required for full function, adding an additional site at different locations within non-conserved sequences interferes with enhancer activity. Of particular note, none of the mutations resulted in ectopic reporter expression outside of the early NB expression window, suggesting that the temporally restricted pattern is defined by transcriptional activators and not by direct DNA binding repressors. Our work also points to an unexpectedly large number of TFs required for optimal enhancer function - mutant TF analysis has identified at least four that are required for full enhancer regulation.
Mechanisms of development 02/2011; 128(3-4):165-77. · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have identified clusters of conserved sequences constituting discrete modular enhancers within the Drosophilanerfin-1 locus. nerfin-1 encodes a Zn-finger transcription factor that directs pioneer interneuron axon guidance. nerfin-1 mRNA is detected in many early delaminating neuroblasts, ganglion mother cells and transiently in nascent neurons. The comparative genomics analysis program EvoPrinter revealed conserved sequence blocks both upstream and downstream of the transcribed region. By using the aligning regions of different drosophilids as the reference DNA, EvoPrinter detects sequence length flexibility between clusters of conserved sequences and thus facilitates differentiation between closely associated modular enhancers. Expression analysis of enhancer-reporter transgenes identified enhancers that drive expression in different regions of the developing embryonic and adult nervous system, including subsets of embryonic CNS neuroblasts, GMCs, neurons and PNS neurons. In summary, EvoPrinter facilitates the discovery and analysis of enhancers that control crucial aspects of nerfin-1 expression.
[show abstract][hide abstract] ABSTRACT: Multi-genome comparative analysis has yielded important insights into the molecular details of gene regulation. We have developed EvoPrinter, a web-accessed genomics tool that provides a single uninterrupted view of conserved sequences as they appear in a species of interest. An EvoPrint reveals with near base-pair resolution those sequences that are essential for gene function.
We describe here EvoPrinterHD, a 2nd-generation comparative genomics tool that automatically generates from a single input sequence an enhanced view of sequence conservation between evolutionarily distant species. Currently available for 5 nematode, 3 mosquito, 12 Drosophila, 20 vertebrate, 17 Staphylococcus and 20 enteric bacteria genomes, EvoPrinterHD employs a modified BLAT algorithm [enhanced-BLAT (eBLAT)], which detects up to 75% more conserved bases than identified by the BLAT alignments used in the earlier EvoPrinter program. The new program also identifies conserved sequences within rearranged DNA, highlights repetitive DNA, and detects sequencing gaps. EvoPrinterHD currently holds over 112 billion bp of indexed genomes in memory and has the flexibility of selecting a subset of genomes for analysis. An EvoDifferences profile is also generated to portray conserved sequences that are uniquely lost in any one of the orthologs. Finally, EvoPrinterHD incorporates options that allow for (1) re-initiation of the analysis using a different genome's aligning region as the reference DNA to detect species-specific changes in less-conserved regions, (2) rapid extraction and curation of conserved sequences, and (3) for bacteria, identifies unique or uniquely shared sequences present in subsets of genomes.
EvoPrinterHD is a fast, high-resolution comparative genomics tool that automatically generates an uninterrupted species-centric view of sequence conservation and enables the discovery of conserved sequences within rearranged DNA. When combined with cis-Decoder, a program that discovers sequence elements shared among tissue specific enhancers, EvoPrinterHD facilitates the analysis of conserved sequences that are essential for coordinate gene regulation.
[show abstract][hide abstract] ABSTRACT: Acquisition of virulence factors and antibiotic resistance by many clinically important bacteria can be traced to horizontal gene transfer (HGT) between related or evolutionarily distant microflora. Comparative genomic analysis has become an important tool for identifying HGT DNA in emerging pathogens. We have adapted the multi-genome alignment tool EvoPrinter to facilitate discovery of HGT DNA sequences within bacterial genomes and within their mobile genetic elements.
EvoPrinter analysis of 13 different Staphylococcus aureus genomes revealed that one of the human isolates, the hospital epidemic methicillin-resistant MRSA252 strain, uniquely shares multiple putative HGT DNA sequences with different causative agents of bovine mastitis that are not found in the other human S. aureus isolates. MRSA252 shares over 14 different DNA sequence blocks with the bovine mastitis ET3 S. aureus strain RF122, and many of the HGT DNAs encode virulence factors. EvoPrinter analysis of the MRSA252 chromosome also uncovered virulence-factor encoding HGT events with the genome of Listeria monocytogenes and a Staphylococcus saprophyticus associated plasmid. Both bacteria are also causal agents of contagious bovine mastitis.
EvoPrinter analysis reveals that the human MRSA252 strain uniquely shares multiple DNA sequence blocks with different causative agents of bovine mastitis, suggesting that HGT events may be occurring between these pathogens. These findings have important implications with regard to animal husbandry practices that inadvertently enhance the contact of human and livestock bacterial pathogens.
PLoS ONE 02/2008; 3(8):e3074. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers.
Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs) made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS.
The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.
[show abstract][hide abstract] ABSTRACT: The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter-controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, we have studied their ability to block or limit the expression of reporter protein in nerfin-1-expressing cells during embryonic development. Our results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression.
[show abstract][hide abstract] ABSTRACT: A systematic approach is described for analysis of evolutionarily conserved cis-regulatory DNA using cis-Decoder, a tool for discovery of conserved sequence elements that are shared between similarly regulated enhancers. Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements.
[show abstract][hide abstract] ABSTRACT: One of the major goals of neurobiology is to describe, in molecular terms, how a neural progenitor cell can generate an ordered series of uniquely fated neurons and glia. It has become clear that many, or all, neural-subtype identities can be linked to sequentially changing regulatory programs within neural precursors. Recent studies shed light on regulatory inputs and timing mechanisms that generate temporally defined cell identities, and new contributions are beginning to establish a link between the temporal network and cell function.
Current Opinion in Cell Biology 01/2006; 17(6):672-5. · 11.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Here, we describe a multigenomic DNA sequence-analysis tool, evoprinter, that facilitates the rapid identification of evolutionary conserved sequences within the context of a single species. The evoprinter output identifies multispecies-conserved DNA sequences as they exist in a reference DNA. This identification is accomplished by superimposing multiple reference DNA vs. test-genome pairwise blat (blast-like alignment tool) readouts of the reference DNA to identify conserved nucleotides that are shared by all orthologous DNAs. evoprinter analysis of well characterized genes reveals that most, if not all, of the conserved sequences are essential for gene function. For example, analysis of orthologous genes that are shared by many vertebrates identifies conserved DNA in both protein-encoding sequences and noncoding cis-regulatory regions, including enhancers and mRNA microRNA binding sites. In Drosophila, the combined mutational histories of five or more species affords near-base pair resolution of conserved transcription factor DNA-binding sites, and essential amino acids are revealed by the nucleotide flexibility of their codon-wobble position(s). Conserved small peptide-encoding genes, which had been undetected by conventional gene-prediction algorithms, are identified by the codon-wobble signatures of invariant amino acids. Also, evoprinter allows one to assess the degree of evolutionary divergence between orthologous DNAs by highlighting differences between a selected species and the other test species.
Proceedings of the National Academy of Sciences 11/2005; 102(41):14700-5. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many studies have focused on the mechanisms of axon guidance; however, little is known about the transcriptional control of the navigational components that carryout these decisions. This report describes the functional analysis of Nerfin-1, a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. We show that the neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development nor in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Our studies also show that Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oalpha47A, Lar, and futsch, genes whose encoded proteins participate in these early navigational events.
[show abstract][hide abstract] ABSTRACT: The goal of neurogenetics is an understanding of the genetic basis of brain structure and function. Neurogenetics deals with multiple areas of investigation, including the genetic basis of neural induction, patterning, cell fate specification, neuron maturation, axonal and dendritic organization, synapse function, and learning and behavior. This appendix provides links to databases and other Web sites used by neurobiologists for recovery of information about genes involved in neural development and neuron function. Special care has been taken to curate sites involving model organisms, since neurogenetics relies heavily on comparative genomics to establish gene function.
Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.] 10/2004; Appendix 5:Appendix 5B.
[show abstract][hide abstract] ABSTRACT: This article considers the evidence for temporal transitions in CNS neural precursor cell gene expression during development. In Drosophila, five prospective competence states have so far been identified, characterized by the successive expression of Hb-->Kr-->Pdm-->Cas-->Gh in many, but not all, neuroblasts. In each temporal window of transcription factor expression, the neuroblast generates sublineages whose temporal identity is determined by the competence state of the neuroblast at the time of birth of the sublineage. Although similar regulatory programs have not yet been identified in mammals, candidate regulatory genes have been identified. Further investigation of the genetic programs that guide both invertebrate and vertebrate neural precursor cell lineage development will ultimately lead to an understanding of the molecular events that control neuronal diversity.
Development 08/2002; 129(16):3763-70. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: During Drosophila neuroblast lineage development, temporally ordered transitions in neuroblast gene expression have been shown to accompany the changing repertoire of functionally diverse cells generated by neuroblasts. To broaden our understanding of the biological significance of these ordered transitions in neuroblast gene expression and the events that regulate them, additional genes have been sought that participate in the timing and execution of these temporally controlled events. To identify dynamically expressed neural precursor genes, we have performed a differential cDNA hybridization screen on a stage specific embryonic head cDNA library, followed by whole-mount embryo in situ hybridizations. Described here are the embryonic expression profiles of 57 developmentally regulated neural precursor genes. Information about 2389 additional genes identified in this screen, including 1614 uncharacterized genes, is available on-line at 'BrainGenes: a search for Drosophila neural precursor genes' (http://sdb.bio.purdue.edu/fly/brain/ahome.htm).
Mechanisms of Development 05/2002; 113(1):41-59. · 2.38 Impact Factor