Conserved Noncoding Sequences Research Articles

Evolution of developmental regulation in the vertebrate FgfD subfamily

Fibroblast growth factors (Fgfs) encode small signaling proteins that help regulate embryo patterning. Fgfs fall into seven families, including FgfD. Nonvertebrate chordates have a single FgfD gene; mammals have three (Fgf8, Fgf17, and Fgf18); and teleosts have six (fgf8a, fgf8b, fgf17, fgf18a, fgf18b, and fgf24). What are the evolutionary processes that led to the structural duplication and functional diversification of FgfD genes during vertebrate phylogeny? To study this question, we investigated conserved syntenies, patterns of gene expression, and the distribution of conserved noncoding elements (CNEs) in FgfD genes of stickleback and zebrafish, and compared them with data from cephalochordates, urochordates, and mammals. Genomic analysis suggests that Fgf8, Fgf17, Fgf18, and Fgf24 arose in two rounds of whole genome duplication at the base of the vertebrate radiation; that fgf8 and fgf18 duplications occurred at the base of the teleost radiation; and that Fgf24 is an ohnolog that was lost in the mammalian lineage. Expression analysis suggests that ancestral subfunctions partitioned between gene duplicates and points to the evolution of novel expression domains. Analysis of CNEs, at least some of which are candidate regulatory elements, suggests that ancestral CNEs partitioned between gene duplicates. These results help explain the evolutionary pathways by which the developmentally important family of FgfD molecules arose and the deduced principles that guided FgfD evolution are likely applicable to the evolution of developmental regulation in many vertebrate multigene families.

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.

C-Myb and GATA-3 Cooperatively Regulate Th2 Cytokine Gene Expression, with the Most Profound Effect On IL-13.

Abstract 3669Poster Board III-605The c-myb proto-oncogene encodes a transcription factor, c-Myb, which regulates genes important for early myeloid, B, and T lymphoid cell development. Our group has recently shown that c-Myb binds a conserved site in the GATA-3 promoter, upregulating its expression and forming a complex that plays an important role in human Th2 cell development. We have also observed that silencing c-Myb in primary human peripheral blood CD4+ cells under Th2 cell promoting conditions decreases the expression of the Th2 cytokines, IL-4, IL-5 and IL-13. We wondered if this was a direct effect of c-Myb alone, or was also due to an effect of the c-Myb/GATA-3 complex? The human IL-4, IL-5 and IL-13 genes are located within a 150kb locus that is called the “Th2 cytokine gene” on Chromosome 5. The Th2 cytokine locus has numerous DNase I hypersensitive sites (DHSs) which act as regulatory elements for Th2 cytokine gene expression. Among these DHSs, the conserved GATA-3 response element (CGRE), the conserved noncoding sequence 1 (CNS-1), the intronic enhancer (IE) and the hypersensitive site V (HSV) are all known to be activated by GATA-3 directly. Interestingly, sequence analysis identified c-Myb binding sites in close proximity to GATA binding sites within all 4 DHSs. To determine if c-Myb, and GATA-3, occupied these putative binding sites, we performed ChIP assays and found that anti-c-Myb antibody, for example, enriched the CGRE locus 30-fold compared to control IgG. Similar experiments revealed that both c-Myb and GATA-3 bound to all 4 of the elements in human naïve CD4+ T-cells under Th2 promoting conditions. To determine the role of c-Myb and GATA-3 in Th2 cytokine gene expression, we performed reporter assays in 293T, Jurkat and human CD4+ T-cells using IL-4, IL-13 and IL-5 promoter constructs with, or without, the 4 DHSs. c-Myb weakly (2-2.5-fold), but significantly, activated both the IL-4 and IL-5 promoters in 293T cells in the absence of DHSs. In contrast, GATA-3 did not activate the IL-4 and IL-5 promoters without the enhancer elements, though it weakly activated the IL-4 promoter through CNS-1. GATA-3 activated the minimal IL-13 promoter 5-fold, and the addition of c-Myb increased promoter activation 10-fold. In marked contrast to these results, an IL-13 promoter construct containing the CGRE enhancer was activated 65-fold by GATA-3 alone, and 160-fold by the addition of c-Myb. The CGRE locus has only one canonical Myb binding site, but Myb's strong enhancer effect on IL-13 expression was completely lost when the CGRE element's Myb binding site was mutated. Mutating the GATA binding sites decreased IL-13 promoter activity by ∼50%. These results suggest that c-Myb and GATA-3 regulate IL-13 expression directly, and in a cooperative manner. These results lead us to hypothesize that the binding of a c-Myb and GATA-3 complex to the CGRE element changes the chromatin structure of the IL-13 locus which acts as a switch to promote IL-13 expression. Though the most dramatic effects of c-Myb were on IL-13 expression, because c-Myb binds to the other three enhancers, and activates both the IL-4 and IL-5 promoters as well, we hypothesize that c-Myb directly contributes to all Th2 cytokine gene expressions, including IL-13. Finally, a ChIP assay showed that MLL also bound to the locus of CGRE. Since we have already shown that c-Myb can bind to MLL through Menin those results suggest, in aggregate, the possibility of histone modification in Th2 cytokine locus by MLL, GATA-3 and c-Myb. This possibility is under active investigation in our laboratory. Disclosures:No relevant conflicts of interest to declare.

A systematic approach to identify functional motifs within vertebrate developmental enhancers

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of approximately 100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.

Melanocyte and RPE‐specific expression in transgenic mice by mouse MART‐1/Melan‐A/mlana regulatory sequences

Figure S1. Identification and analysis of conserved non-coding sequences (CNS) 5′ to the mouse MART-1 gene. Four CNS sequences were analyzed in transient transfections and in transient transgenic embryos. Figure S2. Expression from MART-1(6.0kb)::lacZ transgenic mice. Expression is detected in embryonic eye (RPE) and in melanocytes after birth. Figure S3. MART-1 BAC targeting strategy. Table S1. Luciferase reporter plasmids. Primers and digestions used for generating the different constructs. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Cis-regulation and chromosomal rearrangement of the fgf8 locus after the teleost/tetrapod split

The complex expression pattern of fibroblast growth factor 8 (Fgf8) and the cellular responses dependent on concentration of its mRNA in vertebrates suggest that Fgf8 should be tightly controlled at the transcriptional level. We found zebrafish conserved noncoding elements (CNEs) with pan-vertebrate as well as fish-specific orthologous sequences from across 200 kb of the zebrafish fgf8a genomic regulatory block to direct reporter expression in patterns consistent with the expression pattern of fgf8a. These included elements from inside the introns of the skin-specific slc2a15a and the ubiquitously expressed fbxw4 bystander genes. The fgf8a/fbxw4 gene pair, which has remained joined throughout three whole genome duplications in chordate evolution, is inverted in teleost genomes, but CNEs across both evolutionary breakpoints showed specific activity. While some CNEs directed highly reproducible expression patterns, others were subject to variation but showed, in a subset of transgenes, expression in the apical ectodermal ridge, the anterior boundaries of somites and the midbrain-hindbrain boundary, specific Fgf8 signaling domains, suggesting that their activity may be context specific. A human element with tetrapod-specific orthologous sequences directed reporter expression to the vasculature, possibly corresponding to a tetrapod innovation. We conclude that fgf8a transcriptional regulation employs pan-vertebrate and teleost-specific enhancers dispersed over three genes in the zebrafish genome.

Elephant shark ( Callorhinchus milii ) provides insights into the evolution of Hox gene clusters in gnathostomes

We have sequenced and analyzed Hox gene clusters from elephant shark, a holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with 45 Hox genes that include orthologs for a higher number of ancient gnathostome Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7 paralogous groups that contain all of the 4 members indicated an ((AB)(CD)) topology for the order of Hox cluster duplication, providing support for the 2R hypothesis (i.e., 2 rounds of whole-genome duplication during the early evolution of vertebrates). Comparisons of noncoding sequences of the elephant shark and human Hox clusters have identified a large number of conserved noncoding elements (CNEs), which represent putative cis-regulatory elements that may be involved in the regulation of Hox genes. Interestingly, in fugu more than 50% of these ancient CNEs have diverged beyond recognition in the duplicated (HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters. Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are indeed enhancers directing tissue-specific expression of Hox genes, divergence of their sequences in vertebrate lineages might have led to altered expression patterns and presumably the functions of their associated Hox genes.

Comparative genomics and evolution of conserved noncoding elements (CNE) in rainbow trout

BackgroundRecent advances in the accumulation of genetic mapping and DNA sequence information from several salmonid species support the long standing view of an autopolyploid origin of these fishes (i.e., 4R). However, the paralogy relationships of the chromosomal segments descendent from earlier polyploidization events (i.e., 2R/3R) largely remain unknown, mainly due to an unbalanced pseudogenization of paralogous genes that were once resident on the ancient duplicated segments. Inter-specific conserved noncoding elements (CNE) might hold the key in identifying these regions, if they are associated with arrays of genes that have been highly conserved in syntenic blocks through evolution. To test this hypothesis, we investigated the chromosomal positions of subset of CNE in the rainbow trout genome using a comparative genomic framework.ResultsThrough a genome wide analysis, we selected 41 pairs of adjacent CNE located on various chromosomes in zebrafish and obtained their intervening, less conserved, sequence information from rainbow trout. We identified 56 distinct fragments corresponding to about 150 Kbp of sequence data that were localized to 67 different chromosomal regions in the rainbow trout genome. The genomic positions of many duplicated CNE provided additional support for some previously suggested homeologies in this species. Additionally, we now propose 40 new potential paralogous affinities by analyzing the variation in the segregation patterns of some multi-copy CNE along with the synteny association comparison using several model vertebrates. Some of these regions appear to carry signatures of the 1R, 2R or 3R duplications. A subset of these CNE markers also demonstrated high utility in identifying homologous chromosomal segments in the genomes of Atlantic salmon and Arctic charr.ConclusionCNE seem to be more efficacious than coding sequences in providing insights into the ancient paralogous affinities within the vertebrate genomes. Such a feature makes these elements extremely attractive for comparative genomics studies, as they can be treated as 'anchor' markers to investigate the association of distally located candidate genes on the homologous genomic segments of closely or distantly related organisms.

Conserved noncoding elements and the evolution of animal body plans

The genomes of vertebrates, flies, and nematodes contain highly conserved noncoding elements (CNEs). CNEs cluster around genes that regulate development, and where tested, they can act as transcriptional enhancers. Within an animal group CNEs are the most conserved sequences but between groups they are normally diverged beyond recognition. Alternative CNEs are, however, associated with an overlapping set of genes that control development in all animals. Here, we discuss the evidence that CNEs are part of the core gene regulatory networks (GRNs) that specify alternative animal body plans. The major animal groups arose >550 million years ago. We propose that the cis-regulatory inputs identified by CNEs arose during the "re-wiring" of regulatory interactions that occurred during early animal evolution. Consequently, different animal groups, with different core GRNs, contain alternative sets of CNEs. Due to the subsequent stability of animal body plans, these core regulatory sequences have been evolving in parallel under strong purifying selection in different animal groups.

Regulation of DNA Demethylation during Maturation of CD4+ Naive T Cells by the Conserved Noncoding Sequence 1

Demethylation of transcriptional regulatory elements and gene coding regions is an important step in the epigenetic regulation of gene expression. Several noncoding conserved regions are required for the efficient transcription of cytokine genes. In this paper, we show that the deletion of one such sequence, conserved noncoding sequence 1 (CNS-1), interferes with the efficient demethylation of Th2 cytokine genes but has little effect on histone modifications in the area. Th2 cells derived from CD4 single-positive (SP) mature thymocytes exhibit more rapid demethylation of CNS-1 and Th2-specific cytokine genes and produce more Th2 cytokines than do Th2 cells derived from CD4-positive peripheral naive T cells. De-repression of the Th1 cytokine IFN-gamma was also detected in Th2-primed CD4 SP thymocytes but not in naive T cells. Our results indicate that susceptibility to demethylation determines the efficiency and kinetics of cytokine gene transcription. The extrathymic maturation step undergone by naive T cells suppresses robust and rapid cytokine expression, whereas mature CD4 SP thymocytes maintain a rapid and less-specific cytokine expression profile. Finally, we detected the methyl cytosine binding protein MBD2 at CNS-1 in mature thymocytes, suggesting that this protein may regulate the demethylation of this region.

The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2

Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.

A cluster of three long-range enhancers directs regionalShhexpression in the epithelial linings

The sonic hedgehog (Shh) pathway plays indispensable roles in the morphogenesis of mouse epithelial linings of the oral cavity and respiratory and digestive tubes. However, no enhancers that regulate regional Shh expression within the epithelial linings have been identified so far. In this study, comparison of genomic sequences across mammalian species and teleost fishes revealed three novel conserved non-coding sequences (CNCSs) that cluster in a region 600 to 900 kb upstream of the transcriptional start site of the mouse Shh gene. These CNCSs drive regional transgenic lacZ reporter expression in the epithelial lining of the oral cavity, pharynx, lung and gut. Together, these enhancers recapitulate the endogenous Shh expression domain within the major epithelial linings. Notably, genomic arrangement of the three CNCSs shows co-linearity that mirrors the order of the epithelial expression domains along the anteroposterior body axis. The results suggest that the three CNCSs are epithelial lining-specific long-range Shh enhancers, and that their actions partition the continuous epithelial linings into three domains: ectoderm-derived oral cavity, endoderm-derived pharynx, and respiratory and digestive tubes of the mouse. Targeted deletion of the pharyngeal epithelium specific CNCS results in loss of endogenous Shh expression in the pharynx and postnatal lethality owing to hypoplasia of the soft palate, epiglottis and arytenoid. Thus, this long-range enhancer is indispensable for morphogenesis of the pharyngeal apparatus.

Conserved noncoding sequences (CNSs) in higher plants

Plant conserved noncoding sequences (CNSs)--a specific category of phylogenetic footprint--have been shown experimentally to function. No plant CNS is conserved to the extent that ultraconserved noncoding sequences are conserved in vertebrates. Plant CNSs are enriched in known transcription factor or other cis-acting binding sites, and are usually clustered around genes. Genes that encode transcription factors and/or those that respond to stimuli are particularly CNS-rich. Only rarely could this function involve small RNA binding. Some transcribed CNSs encode short translation products as a form of negative control. Approximately 4% of Arabidopsis gene content is estimated to be both CNS-rich and occupies a relatively long stretch of chromosome: Bigfoot genes (long phylogenetic footprints). We discuss a 'DNA-templated protein assembly' idea that might help explain Bigfoot gene CNSs.

Deletion of a conserved noncoding sequence in Plzf intron leads to Plzf down‐regulation in limb bud and polydactyly in the rat

Lx mutation in SHR.Lx rat manifests in homozygotes as hindlimb preaxial polydactyly. It was previously mapped to a chromosome 8 segment containing the Plzf gene. Plzf (promyelocytic leukemia zinc finger protein) influences limb development as a direct repressor of posterior HoxD genes. However, the Plzf coding sequence is intact in the Lx mutants. Using linkage mapping in F2 hybrids, we downsized the segment containing Lx to 155 kb and sequenced conserved noncoding elements (CNEs) inside. A 2,964-bp deletion in Plzf intron 2, never detected in control animals, is the only candidate for Lx. The deletion removes the most deeply conserved CNE in the 155-kb segment, suggesting a regulatory influence on Plzf expression. Correspondingly, using in situ hybridization and quantitative real-time polymerase chain reaction, we found a decrease of Plzf expression in Lx/Lx limb buds with concomitant anterior expansion of expression domains of its targets, Hoxd10-13 genes, in the absence of ectopic Sonic hedgehog expression. Upstream regulation of Plzf in limb buds is currently unknown. We present here the first candidate Plzf cis-regulatory sequence.

Transcriptional Regulation of Gata3 in T Helper Cells by the Integrated Activities of Transcription Factors Downstream of the Interleukin-4 Receptor and T Cell Receptor

GATA3 is a critical transcription factor for many developmental processes. During T helper (Th) cell differentiation, GATA3 induces the Th2 and suppresses the Th1 pathway. Stimulation of the T cell receptor (TCR) of naive Th cells in the presence of interleukin 4 (IL-4) induces robust expression of GATA3; however, it is unclear where these signals integrate. Gata3 encodes two transcripts that differ in their alternative, untranslated first exons. We show here the involvement of the TCR-inducible transcription factor NFAT1 in the transcriptional regulation of both Gata3 transcripts following TCR stimulation of naive and differentiated Th2 cells. We also show that IL-4 is important for the initiation and establishment of Gata3 transcription in developing Th2 cells, especially from the distal promoter. The early function of IL-4 can be STAT6 dependent or independent. However, the establishment of the activity of the distal promoter is totally dependent on STAT6, whereas it is likely that the proximal promoter has additional activation mechanisms that are STAT6 independent. Our findings suggest that different combinations of transcription factors downstream of the IL-4 receptor (IL-4R) and TCR finely modulate Gata3 gene expression from its two promoters for optimal Th2 differentiation.

Characterization and evolutionary landscape of AmnSINE1 in Amniota genomes

Discovery of a large number of conserved non-coding elements (CNEs) in vertebrate genomes provides a cornerstone to elucidate molecular mechanisms of macroevolution. Extensive comparative genomics has proven that transposons such as short interspersed elements (SINEs) were an important source of CNEs. We recently characterized AmnSINE1, a SINE family in Amniota genomes, some of which are present in CNEs, and demonstrated that two AmnSINE1 loci play an important role in mammalian-specific brain development by functioning as an enhancer (Sasaki et al. Proc. Natl. Acad. Sci. USA 2008). To get more information about AmnSINE1s, we here performed a multi-species search for AmnSINE1, and revealed the distribution and evolutionary history of these SINEs in amniote genomes. The number of AmnSINE1 regions in amniotes ranged from 160 to 1200; the number in the eutherians were under 500 and the largest was that in chicken. Phylogenetic analysis established that each AmnSINE1 locus has evolved uniquely, primarily since the divergence of mammals from reptiles. These results support the notion that AmnSINE1s were amplified as an ancient retroposon in a common ancestor of Amniota and subsequently have survived for 300 Myr because of functions acquired by mutation-coupled exaptation prior mammalian radiation. On the basis of sequence homology and conserved synteny, we detected the orthologs of AmnSINE1 for candidates of further enhancer analysis, which are more conserved than two loci that were shown to have been involved in mammalian brain development. The present work provides a comprehensive data set to test the role of AmnSINE1s, many of which were exapted and contributed to mammalian macroevolution.

Mutation analysis of a large Chinese pedigree with congenital preaxial polydactyly

Mutations in the long-range limb-specific cis-regulator (ZRS) could cause ectopic shh gene expression and are responsible for preaxial polydactyly (PPD). In this study, we analyzed a large Chinese isolated autosomal dominant PPD pedigree. By fine mapping and haplotype construction, we located the linked region to a 1.7 cM interval between flanking markers D7S2465 and D7S2423 of chromosome 7q36. We directly sequenced the candidate loci in this linked region, including the coding regions of the five genes (HLXB9, LMBR1, NOM1, RNF32 and C7orf13), the regulatory element (ZRS) of shh, the whole intron 5 of LMBR1 which contained the ZRS, and 18 conserved noncoding sequences (CNSs). Interestingly, no pathogenic mutation was identified. By using real-time quantitative PCR (qPCR), we also excluded the ZRS duplication in this pedigree. Our results indicate that, at least, it is not the mutation in a functional gene, CNS region or duplication of ZRS that cause the phenotype of this pedigree. The etiology of this PPD family still remains unclear and the question whether another limb-specific regulatory element of shh gene exists in the noncoding region in this 1.7 cM interval remains open for future research.

Recurrent Chromosomal Intermingling Interactions at the BCL2 Locus in T(14;18) +Ve and −Ve Cell Lines.

Introduction Chromosomes locate non-randomly within the nucleus where they physically associate via intermingling. This physical association leads to co-ordinated regulation of gene expression but also makes these regions prone to translocations. The loci involved in many of the common translocations in lymphoma co-localise together and include IGH and BCL2 which are juxtaposed by t(14,18)(q32q21) in ~90% of Follicular Lymphoma (FL). This frequency suggests the BCL2 locus may be prone to intermingling, with the major breakpoint region (MBR) representing a potential interactive hotspot as it flanks a matrix attachment region (MAR) which tethers chromatin.Methods-Results To investigate this possibility, intermingling associations with the BCL2 locus were determined using the Chromosome Conformation Capture technique (3C) in B-lympho-blastoid (HRC57 and NcNc), t(14;18) +ve (RL, SuDHL4, SuDHL6, DoHH2) and -ve (HT, SuDHL8, SuDHL5, RAJI) B-NHL cell lines. The 3C technique allows cross-linking of associating chromosomal segments, generating a library of DNADNA hybrid molecules. Intermingling profiles were established for the following three BCL2 fragments contained within this library:a 10kb section containing the BCL2 promoter region including exons 1 and 2;a 7kb segment within intron 2 containing a highly Conserved Non-coding Sequence (CNS) (associated with gene regulation) and overlapping MAR; anda 6kb section containing part of exon 3 and the MBR with flanking MAR.This selection contained 81% of the BCL2 coding sequence. The intermingling partners of these BCL2 fragments were amplified using either BCL2- “unknown DNA walking primer” or BCL2-IGH enhancer primer combinations and their identity determined by direct sequencing. Nine intermingling partners were identified from the 3C libraries of at least two cell lines. Of these, IGH enhancer–BCL2 promoter associations were identified in RL, SuDHL4 and DoHH2 and appear restricted to t(14;18)+ve cell lines. MYCBP2 (chr13q22), which functions as a transcriptional activator of MYC, associated with the CNS+MAR sequence within intron 2 and this association was detected solely in malignant cell lines. In comparison, OPCML (chr11q25), a tumour-related gene, associated with the BCL2 MBR+MAR region in all cell lines examined.Conclusions These preliminary studies suggest that the BCL2 locus has multiple recurring intermingling partners. These may be specific to BCL2, as demonstrated for OPCML which was a consistent partner in all lines investigated, or may be restricted to cells containing the translocated allele as shown for the IGH enhancer.

Isolation and characterization of conserved non-coding sequences among rice (Oryza sativa L.) paralogous regions

Segmental duplication is particularly frequent within plant genomes and the ability of the original single-copy gene to gain a new function for the change of regulatory elements is one of the prominent consequences of duplication. Thus, it is important to study the pattern of conserved non-coding sequence (CNS) between paralogous genes. We report the result of a survey of CNSs among paralogous regions in rice (Oryza sativa L.), as well as the comparison of CNS dataset between rice and Arabidopsis thaliana. Some common properties, such as the change of A + T content near the CNS boundaries and CNS are enriched in regulatory genes, were observed. However, the content of CNSs differs between rice and Arabidopsis, and it is interesting that the rice metabolic network includes both CNS-poor and CNS-rich genes, which indicated a fine-tuned metabolic network presents in rice.

CneViewer: a database of conserved non-coding elements for studies of tissue-specific gene regulation

There are thousands of strongly conserved non-coding elements (CNEs) in vertebrate genomes, and their functions remain largely unknown. However, without biologically relevant criteria for prioritizing them, selecting a 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 here 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.