Cis Regulatory Elements

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence. D

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116 272 ESTs were produced, comprising 100 674 5′ ESTs and 15 598 3′ ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18 876 contigs and 23 034 singletons, or 41 910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the1536 contigs that contained at least 10 5′ EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.

We have used in situ hybridization to analyze Na,K-ATPase a and b subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All a and b subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five a1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The a2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two a3 genes are restricted in their expression to the nervous system. Of the six b subunit genes, b1a is expressed at highest abundance in lens, pronephros, and heart, while b1b transcripts are abundant in mucous cells. The two b2-like genes are differentially expressed in the nervous system. One b3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 a/b subunit pairs are likely to be formed in different tissues. q

This paper describes a novel approach to constructing Position-Specific Weight Matrices (PWMs) based on the transcription factor binding site (TFBS) data provide by the TRANSFAC database and comparison of the newly generated PWMs with the original TRANSFAC matrices. Multiple local sequence alignment was performed on the TFBSs of each transcription factor. Several different alignment programs were tested and their matrices were compared to the original TRANSFAC matrices. One of the alignment programs, GLAM, produced comparable matrices in terms of the average ranking of true positive sites across the whole test set of sequences.

Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences.

Plants are sessile and therefore exposed to a number of biotic and abiotic stresses. Drought is the major abiotic stress restricting plant growth worldwide. A number of genes involved in drought stress response have already been characterized, mainly in the model species Arabidopsis thaliana and Oryza sativa. However, with the aim to produce drought tolerant crop varieties, it is of importance to identify the respective orthologs for each species. We have developed DroughtDB, a manually curated compilation of molecularly characterized genes that are involved in drought stress response. DroughtDB includes information about the originally identified gene, its physiological and/or molecular function and mutant phenotypes and provides detailed information about computed orthologous genes in nine model and crop plant species including maize and barley. All identified orthologs are interlinked with the respective reference entry in MIPS/PGSB PlantsDB, which allows retrieval of additional ...

Background: While hundreds of genes have been implicated already in the etiology of schizophrenia, the exact cause is not known or the disease is considered multigenic in origin. Recent discoveries of new types of RNAs and the gradual elimination of the “junk DNA” hypothesis refocused the attention on the noncoding part of the human genome. Here we re-analyzed a recent dataset of differentially methylated genes from schizophrenic patients and cross-tabulated them with cis regulatory and repetitive elements and microRNAs known to be involved in schizophrenia.
Results: We found that the number of schizophrenia-related (SZ) microRNA targets follows a scale-free distribution with several microRNA hubs and that schizophrenia-related microRNAs with shared targets form a small-world network. The top ten microRNAs with the highest number of SZ gene targets regulate approximately 80 % of all microRNA-regulated genes whereas the top two microRNAs regulate 40–52 % of all such genes. We also found that genes that are regulated by the same microRNAs tend to have more protein-protein interactions than randomly selected schizophrenia genes. This highlights the role microRNAs possibly play in coordinating the abundance of interacting proteins, an important function that has not been sufficiently explored before. The analysis revealed that GABBR1 is regulated by both of the top two microRNAs and acts as a hub by interacting with many schizophrenia-related genes and sharing several types of transcription-binding sites with its interactors. We also found that differentially methylated repetitive elements are significantly more methylated in schizophrenia, pointing out their potential role in the disease.
Conclusions: We find that GABBR1 has a central importance in schizophrenia, even if no direct cause and effect have been shown for it for the time. In addition to being a hub in microRNA-derived regulatory pathways and protein-protein interactions, its centrality is also supported by the high number of cis regulatory elements and transcription factor-binding sites that regulate its transcription. These findings are in line with several genome-wide association studies that repeatedly find the major histocompatibility region (where GABBR1 is located) to have the highest number of single nucleotide polymorphisms in schizophrenics. Our model also offers an explanation for the downregulation of protein kinase B, another consistent finding in schizophrenic patients. Our observations support the notion that microRNAs fine-tune the amount of proteins acting in the same biological pathways in schizophrenia, giving further support to the emerging theory of competing endogenous RNAs.
Open Peer Review: The manuscript was reviewed by Jaap Heringa, Sandor Pongor and Zoltan Gaspari. Keywords: Schizophrenia, Methylation, GABBR1, AKT1, MicroRNAs, Gene networks, Protein-protein interactions

Background: Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the ligninspecific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters.

Methionine adenosyltransferase is a ubiquitous enzyme
that catalyzes the only known route of biosynthesis
of S-adenosylmethionine, the major methyl group
donor in cell metabolism. In mammals, two different
methionine adenosyltransferases exist: one is confined
to the liver, and the other one is distributed in extrahepatic
tissues. In the present study, we report the cloning
of the 5*-flanking region of liver-specific methionine adenosyltransferase
gene from rat. Two closely spaced
sites for transcriptional initiation were identified by
primer extension analysis. The major transcription start
site was determined to be 29 nucleotides downstream
from the putative TATA box. Transient transfection assays
of constructs containing sequentially deleted 5*-
flanking sequences fused to the luciferase gene showed
that rat hepatic methionine adenosyltransferase promoter
was able to efficiently drive reporter expression
not only in liver-type cells (rat hepatoma H35 cells and
human hepatoblastoma HepG2 cells) but also in Chinese
hamster ovary cells. Two regions spanning nucleotides
21251 to 2958 and 2197 to 165 were found to be crucial
for the promoter efficiency. The distal upstream region
contains elements that positively regulate promoter activity
in H35 and HepG2 cells but are ineffective in Chinese
hamster ovary cells. Eight protein binding sites
were characterized in both regions by DNase I footprinting
analysis. Two of these elements, sites A and B, located
in the distal region, were found to be essential for
the regulation of promoter activity. Electrophoretic mobility
shift assays and competition experiments showed
that site A is recognized by an NF1 protein. Site B was
able to interact with a member of HNF-3 family when
nuclear extracts from rat liver and H35 cells were used
in the in vitro assay, but an additional binding activity
to an NHF1-like protein was obtained with the hepatoma
cell extracts. It is suggested that this differential
binding can contribute to the cell specificity of promoter
function.

Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes. We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole s...

The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fle...

Enhancers lie at the heart of transcriptional and developmental gene regulation. Therefore, changes in enhancer sequences usually
disrupt the target gene expression and result in disease phenotypes. Despite the well-established role of enhancers in development
and disease, evolutionary sequence studies are lacking. The current study attempts to unravel the puzzle of bony vertebrates’
conserved noncoding elements (CNE) enhancer evolution. Bayesian phylogenetics of enhancer sequences spotlights promising
interordinal relationships among placental mammals, proposing a closer relationship between humans and laurasiatherians while
placing rodents at the basal position. Clock-based estimates of enhancer evolution provided a dynamic picture of interspecific rate
changes across the bony vertebrate lineage. Moreover, coelacanth in the study augmented our appreciation of the vertebrate cis regulatory
evolution during water–land transition. Intriguingly, we observed a pronounced upsurge in enhancer evolution in land dwelling
vertebrates. These novel findings triggered us to further investigate the evolutionary trend of coding as well as CNE
non enhancer repertoires, to highlight the relative evolutionary dynamics of diverse genomic landscapes. Surprisingly, the evolutionary
rates of enhancer sequences were clearly at odds with those of the coding and the CNE non enhancer sequences during vertebrate
adaptation to land, with land vertebrates exhibiting significantly reduced rates of coding sequence evolution in comparison to their
fast evolving regulatory landscape. The observed variation in tetrapod cis-regulatory elements caused the fine-tuning of associated
gene regulatory networks. Therefore, the increased evolutionary rate of tetrapods’ enhancer sequences might be responsible for the
variation in developmental regulatory circuits during the process of vertebrate adaptation to land.

We present the webserver 3D transcription factor (3DTF) to compute position-specific weight matrices (PWMs) of transcription factors using a knowledge-based statistical potential derived from crystallographic data on protein-DNA complexes. Analysis of available structures that can be used to construct PWMs shows that there are hundreds of 3D structures from which PWMs could be derived, as well as thousands of proteins homologous to these. Therefore, we created 3DTF, which delivers binding matrices given the experimental or modeled protein-DNA complex. The webserver can be used by biologists to derive novel PWMs for transcription factors lacking known binding sites and is freely accessible at

In recent years we have witnessed an explosion in our understanding of how genes regulate developmental and physiological processes, largely based on work from a few model genetic organisms. The zebrafish is the newest of these model organisms. Powerful techniques allow efficient generation and recovery of zebrafish mutations affecting genes that regulate developmental patterning, organogenesis, physiology and behavior, and the transparency of zebrafish embryos aids cellular analysis of gene function.

Understanding the mechanisms that govern the expression of genomes is one of the major challenges of the post-genomic era. Phylogenetic footprinting, which identifies genomic regions under evolutionary constraints, has proven helpful in finding cis-regulatory elements of transcription; however, this method may not be applicable across all evolutionary distances and for all types of genes. Recent results from vertebrate comparisons indicate that strong conservation of cis-regulatory regions may occur more frequently in developmental regulator genes. This paper reviews methods of identifying conserved regulatory elements of developmental genes by comparative genomics, including new attempts to detect conserved features beyond simple sequence similarities. The results obtained are outlined and the authors comment on their functional and evolutionary implications. Finally, an evaluation of currently available methods of characterising the function of presumed conserved regulatory regions is presented, and problems such as promoter compatibility, assigning distant elements to their cognate genes and multifunctionality of elements, discussed. Fugu (Takifugu rubripes) http://genome.jgi-psf.org/fugu6/fugu6.home.html , http://www.ensembl.org/Fugu_rubripes/ Green pufferfish (Tetraodon nigroviridis)

Background: All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development.

There are thousands of strongly conserved noncoding elements (CNEs) in vertebrate genomes, and their functions remain largely unknown. However, without biologically relevant criteria for prioritizing them, selecting particular CNE sequences to study can be haphazard. To address this problem, we present cneViewer -a database and webtool that systematizes information on conserved non-coding DNA elements in zebrafish. A key feature is the ability to search for CNEs that may be relevant to tissue-specific gene regulation, based on known developmental expression patterns of nearby genes. cneViewer provides this and other organizing features that significantly facilitate experimental design and CNE analysis.

The binding of some transcription factors has been shown to diverge substantially between closely related species. Here we show that the binding of the developmental transcription factor Twist is highly conserved across six Drosophila species, revealing strong functional constraints at its enhancers. Conserved binding correlates with sequence motifs for Twist and its partners, permitting the de novo discovery of their combinatorial binding. It also includes over 10,000 low-occupancy sites near the detection limit, which tend to mark enhancers of later developmental stages. These results suggest that developmental enhancers can be highly evolutionarily constrained, presumably because of their complex combinatorial nature.

Genes are often regulated in living cells by proteins called transcription factors that bind directly to short segments of DNA in close proximity to specific genes. These binding sites have a conserved nucleotide appearance, which is called a motif. Several recent studies of transcriptional regulation require the reduction of a large collection of motifs into clusters based on the similarity of their nucleotide composition. We present a principled approach to this clustering problem based on a Bayesian hierarchical model that accounts for both within-and between-motif variability. We use a Dirichlet process prior distribution that allows the number of clusters to vary and we also present a novel generalization that allows the core width of each motif to vary. This clustering model is implemented, using a Gibbs sampling strategy, on several collections of transcription factor motif matrices. Our stochastic implementation allows us to examine the variability of our results in addition to focusing on a set of best clusters. Our clustering results identify several motif clusters that suggest that several transcription factor protein families are actually mixtures of several smaller groups of highly similar motifs, which provide substantially more refined information compared with the full set of motifs in the family. Our clusters provide a means by which to organize transcription factors based on binding motif similarities and can be used to reduce motif redundancy within large databases such as JASPAR and TRANSFAC, which aides the use of these databases for further motif discovery. Finally, our clustering procedure has been used in combination with discovery of evolutionarily conserved motifs to predict co-regulated genes. An alternative to our Dirichlet process prior distribution is presented that differs substantially in terms of a priori clustering characteristics, but shows no substantive difference in the clustering results for our dataset. Despite our specific application to transcription factor binding motifs, our Bayesian clustering model based on the Dirichlet process has several advantages over traditional clustering methods that could make our procedure appropriate and useful for many clustering applications.

During Strongylocentrotus purpuratus embryogenesis, aboral ectoderm-specific expression of spec2a relies on an upstream enhancer that confers its spatial specificity largely through repression. The purpose of this study was to determine how spec2a expression is repressed in endoderm and oral ectoderm territories. A 78-base pair DNA sequence within the enhancer contains five tightly spaced cis-regulatory elements including proximal (TAATCT) and distal (TAATCC) elements that bind to both SpOtx, a broadly distributed transcriptional activator, and SpGoosecoid (SpGsc), an oral ectoderm-restricted transcriptional repressor. We show here that these two seemingly redundant Otx/Gsc elements have distinct functions. The proximal element bound to SpGATA-E, an endomesoderm-specific transcription factor. Treatment with SpGATA-E and SpGsc morpholino antisense oligonucleotides (MASOs) resulted in enhanced transcriptional activity from the proximal element, suggesting that both factors functioned as repressors at this site. SpGATA-E MASO-treated embryos failed to express ectoderm markers, indicating a role for SpGATA-E in ectoderm differentiation. The spec2a proximal element was distinct from the corresponding element in the related spec1 enhancer, and swaps between spec1 and spec2a cis-regulatory elements indicated, that for optimal repression, the proximal element had to interact with a nearby CCAAT-binding factor element. Our results show that the recently evolved proximal element contributes to the repression of spec2a in endomesoderm and oral ectoderm territories. D

Transthyretin (TTR)-related amyloidosis is a life-threatening disease. Currently, several questions about the pathogenic mechanisms of TTR-related amyloidosis remain unanswered. We have investigated various TTR-related issues using different in silico approaches. Using an amino acid similarity-based analysis, we have indicated the most relevant TTR secondary structures in determining mutation impact. Our amyloidogenic propensity analysis of TTR missense substitutions has highlighted a similar pattern for wild-type and mutated TTR amino β acid sequences. However, some mutations present differences with respect to the general distribution. We have identified non-coding variants in cis-regulatory elements of the TTR gene, and our analysis on V122I-related haplotypes has indicated differences in non-coding regulatory variants, suggesting differences among V122I carriers. The analysis of methylation status indicated CpG sites that may affect TTR expression. Finally, our interactive netwo...

The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection. q

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [À340/À124] as essential for vascular cambium/ xylem-specific expression. In plants transformed with the smallest promoter region [À124/þ117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [À124/þ117] region contained cis-elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues.

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http:// www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

The model genome of Arabidopsis thaliana contains a DEAD-box RNA helicase family (RH) of 58 members, i.e. almost twice as many as in the animal or yeast genomes. Transcript profiling using real-time quantitative polymerase chain reaction (PCR) has been obtained for 20 AtRHs from nine different organs. Two AtRHs exhibited plant-specific profiles associated with photosynthetic and sink organs. The other 18 AtRHs had the same transcript profile, and the levels of transcription of these ‘housekeeping’AtRHs were under strict quantitative control over a large range of values. Transcript levels may be very different between the most recently duplicated genes. The master regulatory element in the definition of the transcript level is the simultaneous presence of a TATA-box and an intron in the 5′ untranslated region (UTR). There is a positive and highly significant correlation between the size of the 5′ UTR intron and the transcription level, as long as a characteristic TATA-box is present. Our work on the housekeeping AtRHs suggests a scenario for the evolution of duplicated genes, leading to both highly and poorly transcribed genes in the same terminal branch of the phylogenetic tree. The general evolutionary drive of the AtRH family, after duplication of a highly transcribed ancestral AtRH, was towards an alteration of the transcriptional activity of the divergent duplicates through successive events of suppression of the TATA-box and/or the 5′ UTR intron.

The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.

The detection of differentially expressed genes from EST data is of importance for the discovery of potential
biological or pharmaceutical targets, especially when studying biological processes in less characterized organisms and
where large-scale microarrays are not an option. We present a comparison of fi ve different statistical methods for identifying
up-regulated genes through pairwise comparison of EST sets, where one of the sets is generated from a treatment and
the other one serves as a control. In addition, we specifi cally address situations where the sets are relatively small (~2,000–
10,000 ESTs) and may differ in size. The methods were tested on both simulated and experimentally derived data, and
compared to a collection of cold stress induced genes identifi ed by microarrays. We found that combining the method proposed
by Audic and Claverie with Fisher’s exact test and a method based on calculating the difference in relative frequency
was the best combination for maximizing the detection of up-regulated genes. We also introduced the use of a fl exible
cutoff, which takes the size of the EST sets into consideration. This could be considered as an alternative to a static cutoff.
Finally, the detected genes showed a low overlap with those identifi ed by microarrays, which indicates, as in previous studies,
low overall concordance between the two platforms.

The enzyme Cinnamyl Alcohol Dehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5 0 flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Picea glauca (Moench) Voss) and loblolly pine (Pinus taeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P. glauca CAD promoter used P. glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the b-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood.

Cis-regulatory modules (CRMs) and motifs play a central role in tissue and condition-specific gene expression. Here we present Imogene, an ensemble of statistical tools that we have developed to facilitate their identification and implemented in a publicly available software. Starting from a small training set of mammalian or fly CRMs that drive similar gene expression profiles, Imogene determines de novo cis-regulatory motifs that underlie this coexpression. It can then predict on a genome-wide scale other CRMs with a regulatory potential similar to the training set. Imogene bypasses the need of large datasets for statistical analyses by making central use of the information provided by the sequenced genomes of multiple species, based on the developed statistical tools and explicit models for transcription factor binding site evolution. We test Imogene on characterized tissue-specific mouse developmental CRMs. Its ability to identify CRMs with the same specificity based on its de novo created motifs is comparable to that of previously evaluated 'motif-blind' methods. We further show, both in flies and in mammals, that Imogene de novo generated motifs are sufficient to discriminate CRMs related to different developmental programs. Notably, purely relying on sequence data, Imogene performs as well in this discrimination task as a previously reported learning algorithm based on Chromatin Immunoprecipitation (ChIP) data for multiple transcription factors at multiple developmental stages.

Background: Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes. Results: We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity.

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http:// www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http:// www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Clustered Hox genes encode transcription factors that pattern the regional identities of tissues along the anterior-posterior (A-P) axis of animals during embryonic development. They are expressed along the A-P axis collinear with their physical location within a cluster. Several studies have examined the expression of teleost Hox genes from paralog groups (PG) 1-4 in the rhombomeres of the hindbrain and the anterior pharyngeal arches. However, little is known about Hox gene expression within the posterior pharyngeal arches (PA3-7) of teleosts. Here we present the spatio-temporal expression patterns of Hox paralog group (PG) 3-6 in the neural tube and posterior arches of the Japanese medaka (Oryzias latipes). We show that medaka Hox gene expression patterns in the hindbrain are divergent spatially from orthologous genes in mouse and other evolutionarily divergent teleosts, which suggests divergence of cis-regulatory sequences directing hindbrain expression. Further, our study is the first to show the complete teleost Hox PG3-6 expression code in the posterior arches up to the chondrogenic stage of the craniofacial skeletal elements. This study will provide the basis for comparative teleost Hox gene expression profiles in PA3-7 and may help in understanding the mechanisms underlying development of divergent morphological pharyngeal arch skeletal structures among evolutionarily divergent teleosts.

We explored distance preferences in the arrangement of binding motifs for ®ve transcription factors (Bicoid, Kru È ppel, Hunchback, Knirps and Caudal) in a large set of Drosophila cis-regulatory modules (CRMs). Analysis of non-overlapping binding motifs revealed the presence of periodic signals speci®c to particular combinations of binding motifs. The most striking periodic signals (10 bp for Bicoid and 11 bp for Hunchback) suggest preferential positioning of some binding site combinations on the same side of the DNA helix. We also analyzed distance preferences in arrangements of highly correlated overlapping binding motifs, such as Bicoid and Kru È ppel. Based on the distance analysis, we extracted preferential binding site arrangements and proposed models for potential composite elements (CEs) and antagonistic motif pairs involved in the function of developmental CRMs. Our results suggest that there are distinct hierarchical levels in the organization of transcription regulatory information. We discuss the role of the hierarchy in understanding transcriptional regulation and in detection of transcription regulatory regions in genomes.

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http:// www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Je tiens d'abord à remercier mon directeur de thèse, Vincent Hakim, qui m'a procuré un encadrement d'une très grande qualité au cours de ces quatre années passées au Laboratoire de Physique Statistique de l'École Normale Supérieure. Sa disponibilité, son discernement, sa curiosité, son esprit critique, l'étendue de sa culture scientifique dans les disciplines aux modes de réflexion et aux langages si différents que sont la physique fondamentale et la biologie, sa capacité à extraire d'un problème l'essence qui satisfait l'intuition et à chasser le superflu, sa démarche toujours rigoureuse vers le plus testable, le plus vérifiable, le plus dicible, mêlé à une grande humilité intellectuelle en font un chercheur exemplaire qui nourrit l'admiration, et je suis fier d'avoir pu apprendre à ses côtés.

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http:// www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required.

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required.

Freezing tolerance in plants is obtained during a period of low non-freezing temperatures before the winter sets on, through a biological process known as cold acclimation. Cold is one of the major stress factors that limits the growth, productivity and distribution of plants, and understanding the mechanism of cold tolerance is therefore important for crop improvement. Expressed sequence tags (EST) analysis is a powerful, economical and time-efficient way of assembling information on the transcriptome. To date, several EST sets have been generated from cold-induced cDNA libraries from several different plant species. In this study we utilize the variation in the frequency of ESTs sampled from different cold-stressed plant libraries, in order to identify genes preferentially expressed in cold in comparison to a number of control sets. The species included in the comparative study are oat (Avena sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), rice (Oryza sativa) and Arabidopsis thaliana. However, in order to get comparable gene expression estimates across multiple species and data sets, we choose to compare the expression of tentative ortholog groups (TOGs) instead of single genes, as in the normal procedure. We consider TOGs as preferentially expressed if they are detected as differentially expressed by a test statistic and up-regulated in comparison to all control sets, and/or uniquely expressed during cold stress, i.e., not present in any of the control sets. The result of this analysis revealed a diverse representation of genes in the different species. In addition, the derived TOGs mainly represent genes that are long-term highly or moderately expressed in response to cold and/or other stresses.

As increasing numbers of vertebrate genomes are sequenced, comparative genomics offers tremendous promise to unveil mechanisms of transcriptional gene regulation on a large scale. However, the challenge of analysing immense amounts of sequence data and relating primary sequence to function is daunting. Several teleost species occupy crucial niches in the world of comparative genomics, as experimental model organisms of wide utility and living roadmaps of molecular evolution. Extant species have evolved after a teleost-specific genome duplication, and offer the opportunity to examine the evolution of thousands of duplicate gene pairs. Transgenesis in zebrafish is being increasingly employed to functionally examine non-coding sequences, from fish and mammals. Here, we discuss current approaches to the study of gene regulation in teleosts, and the promise of future research.

The detection of differentially expressed genes from EST data is of importance for the discovery of potential biological or pharmaceutical targets, especially when studying biological processes in less characterized organisms and where large-scale microarrays are not an option. We present a comparison of five different statistical methods for identifying up-regulated genes through pairwise comparison of EST sets, where one of the sets is generated from a treatment and the other one serves as a control. In addition, we specifically address situations where the sets are relatively small (micro 2,000-10,000 ESTs) and may differ in size. The methods were tested on both simulated and experimentally derived data, and compared to a collection of cold stress induced genes identified by microarrays. We found that combining the method proposed by Audic and Claverie with Fisher's exact test and a method based on calculating the difference in relative frequency was the best combination for maximizing the detection of up-regulated genes. We also introduced the use of a flexible cutoff, which takes the size of the EST sets into consideration. This could be considered as an alternative to a static cutoff. Finally, the detected genes showed a low overlap with those identified by microarrays, which indicates, as in previous studies, low overall concordance between the two platforms.