Candidate Cis-regulatory Elements Research Articles

Identification of novel cis-regulatory elements of Eya1 in Xenopus laevis using BAC recombineering

The multifunctional Eya1 protein plays important roles during the development of cranial sensory organs and ganglia, kidneys, hypaxial muscles and several other organs in vertebrates. Eya1 is encoded by a complex locus with candidate cis-regulatory elements distributed over a 329 kbp wide genomic region in Xenopus. Consequently, very little is currently known about how expression of Eya1 is controlled by upstream regulators. Here we use a library of Xenopus tropicalis genomic sequences in bacterial artificial chromosomes (BAC) to analyze the genomic region surrounding the Eya1 locus for enhancer activity. We used BAC recombineering to first create GFP reporter constructs, which were analysed for enhancer activity by injection into Xenopus laevis embryos. We then used a second round of BAC recombineering to create deletion constructs of these BAC reporters to localize enhancer activity more precisely. This double recombineering approach allowed us to probe a large genomic region for enhancer activity without assumptions on sequence conservation. Using this approach we were able to identify two novel cis-regulatory regions, which direct Eya1 expression to the somites, pharyngeal pouches, the preplacodal ectoderm (the common precursor region of many cranial sensory organs and ganglia), and other ectodermal domains.

Melatonin Regulates Root Architecture by Modulating Auxin Response in Rice.

It has been suggested that melatonin acts as an important regulator in controlling root growth and development, but the underlying molecular mechanism driving this relationship remains undetermined. In this study, we demonstrated that melatonin acts as a potent molecule to govern root architecture in rice. Treatments with melatonin significantly inhibited embryonic root growth, and promoted lateral root formation and development. Genome-wide expression profiling by RNA-sequencing revealed auxin-related genes were significantly activated under melatonin treatment. Moreover, several transcription factors and candidate cis-regulatory elements involved in root growth and developments, as well as auxin-related processes, were over-represented in both co-up and -down differentially expressed genes, suggesting that melatonin-mediated root growth occurs in an auxin signal pathway-dependent manner. Further, gravitropic response analysis determined that melatonin affects auxin-regulated processes in rice root. These data show that melatonin shapes root architecture by directly or indirectly activating the auxin signaling pathway.

Isolation of a novel leucine-rich repeat receptor-like kinase (OsLRR2) gene from rice and analysis of its relation to abiotic stress responses

ABSTRACTLeucine-rich repeat receptor-like kinase protein (LRR-RLK) is involved in a wide range of biological pathways. So far, the function of LRR-RLK in the growth, developmental processes and various external stimuli has still not been clearly elucidated in rice (Oryza sativa L.). To understand the mechanism(s) underlying stress response and to discover novel stress-tolerance genes in rice, we analysed a global genome expression profiling of the indica cultivar Pei'ai 64S subjected to cold, drought or heat stresses. Expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages from plants under no stress, or cold, drought or heat stress, using the GeneChip Rice Genome Array (Affymetrix) representing 51279 transcripts from japonica and indica rice. We identified a gene, OsLRR2 (Oryza sativa L. leucine-rich repeat receptor-like kinase 2, GenBank accession: EAZ02952.1), which was highly up-regulated under cold and drought stress. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by reverse-transcription polymerase chain reaction. Sequence analysis showed that the cDNA encodes a protein of 375 amino-acid residues with molecular weight of ≈40.62 kD and pI of ≈5.75. The sequence databases search found that the open reading frame of OsLRR2 contained a leucine-rich repeats domain. Analysis of the putative promoter region for candidate cis-regulatory elements identified five matches to cis-elements related to stress responses, suggesting that OsLRR2 could be considered a new candidate gene involved in stress tolerance in rice.

Coordinating the Human Looks

Prescott etal. take a step forward in studying primate morphological evolution by a cellular anthropology approach. Through epigenomic profiling of in-vitro-derived cells, the authors identify and characterize candidate cis-regulatory elements underlying divergence in facial morphology between human and chimp, shedding new light on what makes us (look) human.

Allelome.PRO, a pipeline to define allele-specific genomic features from high-throughput sequencing data.

Detecting allelic biases from high-throughput sequencing data requires an approach that maximises sensitivity while minimizing false positives. Here, we present Allelome.PRO, an automated user-friendly bioinformatics pipeline, which uses high-throughput sequencing data from reciprocal crosses of two genetically distinct mouse strains to detect allele-specific expression and chromatin modifications. Allelome.PRO extends approaches used in previous studies that exclusively analyzed imprinted expression to give a complete picture of the ‘allelome’ by automatically categorising the allelic expression of all genes in a given cell type into imprinted, strain-biased, biallelic or non-informative. Allelome.PRO offers increased sensitivity to analyze lowly expressed transcripts, together with a robust false discovery rate empirically calculated from variation in the sequencing data. We used RNA-seq data from mouse embryonic fibroblasts from F1 reciprocal crosses to determine a biologically relevant allelic ratio cutoff, and define for the first time an entire allelome. Furthermore, we show that Allelome.PRO detects differential enrichment of H3K4me3 over promoters from ChIP-seq data validating the RNA-seq results. This approach can be easily extended to analyze histone marks of active enhancers, or transcription factor binding sites and therefore provides a powerful tool to identify candidate cis regulatory elements genome wide.

Dynamic regulation of mRNA decay during neural development.

BackgroundGene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay.ResultsWe developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.ConclusionsWe found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0038-6) contains supplementary material, which is available to authorized users.

Isolation of a novel abscisic acid stress ripening (OsASR) gene from rice and analysis of the response of this gene to abiotic stresses

Abiotic stresses constitute a serious threat to agricultural production, which often develops into major crop production reducing factors around the world. Molecular biology technology has, however, emerged as a promising vehicle improving crop tolerance. A cold-, drought- and heat-inducible gene designated Oryza sativa L. abscisic acid stress-ripening ( OsASR ) gene, GenBank accession: AK318549.1 was identified in rice Pei’ai64s ( O. sativa L. ssp. Indica cv.) using the GeneChip rice genome array (Affymetrix) representing 51, 279 transcripts from two rice subspecies japonica and indica . The expression profile of OsASR obtained by the microarray analysis was confirmed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of the gene. The two sets of data matched very well, suggesting that OsASR is a multiple stresses responsive gene in rice. Based on the sequence, PCR primers were designed. The cDNA with the whole open reading frame (ORF) was amplified by PCR and cloned. Sequence analysis showed that the cDNA encodes a protein of 284 amino acid residues with M.W. ≈ 11.7 kD and pI ≈ 10.4. The gene encodes a protein with several conserved domains. Comparison of protein sequences indicates that OsASR encodes a putative abscisic acid stress-ripening protein. Analysis of the putative promoter region for candidate cis-regulatory elements using PlantCARE software identified seven kinds of cis-elements related to stress responses. Based on the aforementioned analyses and results obtained, we propose that OsASR is a novel candidate gene involved in stress tolerance in rice. Keywords: Rice, microarray, abiotic stress, reverse transcription polymerase chain reaction (RT-PCR), abscisic acid stress ripening

Multiple cis‐regulatory elements are involved in the complex regulation of the sieve element‐specific MtSEO‐F1 promoter from Medicago truncatula

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.

Isolation of a novel MYB transcription factor OsMyb1R from rice and analysis of the response of this gene to abiotic stresses

To understand mechanisms underlying stress responses and discover new stress-tolerance genes in rice ( Oryza sativa L.), we analyzed a global genome expression profiling of the indica cultivar Pei’ai 64S subjected to cold, drought, or heat stresses. Expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages from plants under no stress, or cold, drought or heat stresses using the GeneChip Rice Genome Array (Affymetrix) representing 51 279 transcripts from japonica and indica rice. A large number of genes highly up- regulated or down-regulated were identified under the stresses. One of these genes, OsMyb1R, was highly induced in leaf and panicle at the heading and flowering stages, in response to all stresses. The expression profile of OsMyb1R obtained by the microarray analysis was confirmed by quantitative real-time reverse transcriptaseplolymerase chain reaction (RT-PCR) analysis of the gene. The two sets of data matched very well, suggesting that OsMyb1R is a multiple stresses responsive gene in rice. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by RT-PCR. Sequence analysis showed that the cDNA encodes a protein of 489 amino acid residues with molecular weight (M.W.) ≈ 56 kD and p I ≈ 6. Bioinformatics analysis revealed that the protein contains a conservative domain of the MYB family gene. Analysis of the putative promoter region for candidate cis-regulatory elements identified 5 matches to cis -elements related to stress responses. Based on the earlier mentioned analysis and results obtained, we propose that OsMyb1R is a novel candidate gene involved in stress tolerance in rice.

Conserved <i>cis</i>-regulatory elements of two odorant-binding protein genes, <i>Obp57d</i> and <i>Obp57e</i>, in <i>Drosophila</i>

Insect odorant-binding proteins function in the sensing of odors, tastes, and pheromones. Genes encoding two odorant-binding proteins, Obp57d and Obp57e, were identified to be involved in the behavioral adaptation of Drosophila sechellia to its host plant. The two genes are expressed in cells associated with taste sensilla on the legs, and the expression pattern in the legs is conserved among closely related species. To identify the cis-regulatory elements necessary for the expression in the leg sensilla, the promoter sequences of Obp57d and Obp57e were compared among species. Two types of conserved sequence-motifs were found as candidate cis-regulatory elements. Functions of these conserved elements in the promoters of D. melanogaster Obp57d and Obp57e were examined by using a newly constructed vector that combines the advantages of φC31 integrase-based transformation and gypsy transposable-element-derived insulators. By GFP-reporter assay using the new vector, it was confirmed that these conserved elements are necessary for the expression in the legs, working synergistically with each other to affect the expression level. Single-nucleotide substitutions in these elements dramatically changed the promoter activity. These results provide insight into the molecular mechanism for evolution of adaptive behavior via modulation of OBP expression levels.

Isolation of a novel xyloglucan endotransglucosylase (OsXET9) gene from rice and analysis of the response of this gene to abiotic stresses

To understand the mechanism(s) underlying stress responses and discover new stress-tolerance genes in rice ( Oryza sativa L.), expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages of indica cultivar Pei’ai 64S plants under cold, drought or heat stresses using the GeneChip Rice Genome Array (Affymetrix) representing 51, 279 transcripts from japonica and indica rice. A large number of genes highly up regulated or down regulated were identified under the stresses. OsXET9 ( O. sativa L. xyloglucan endotransglucosylase 9, GenBank accession: NM_001060322) was highly induced in leaf and panicle at all the developmental stages, in response to all stresses, especially in seedling and booting stage under cold stress. Real-time quantitative PCR analysis showed that the result was consensus with GeneChip Rice Genome Array, suggesting that OsXET9 is a multiple stress responsive gene in rice. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by PCR. Sequence analysis showed that the cDNA encodes a protein of 284 amino acid residues with M.W. ≈ 31kD and pI ≈ 5.2. The gene encodes a protein with several conserved domains. Comparison of protein sequences indicates that OsXET9 is closely related to the xyloglucan endotransglycosylase. Analysis of the putative promoter region for candidate cis-regulatory elements using Plant CARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) identified some cis-elements related to stress responses. Based on these analyses and results obtained, we propose that OsXET9 is a novel candidate gene involved in stress tolerance in rice in addition to the function in metabolism of cell wall. Keywords : Rice, abiotic stress, microarray, xyloglucan endotransglucosylase.

Dynamic usage of alternative splicing exons during mouse retina development

Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements.

A cis-Regulatory Signature for Chordate Anterior Neuroectodermal Genes

One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation.

Identification of the ETS Family Member ELF1 as a Transcriptional Regulator of MEIS1 Expression.

Abstract 3647Poster Board III-583MEIS1 is a Hox cofactor known to be a key regulator of normal hematopoietic and leukemic stem cell function. Overexpression of Meis1 is a potent collaborating event in leukemias associated with multiple Hox, NUP98-HOX and MLL fusion genes. The transcriptional regulation of MEIS1 expression however remains poorly understood. To identify potential transcriptional regulators, we searched for candidate cis-regulatory elements within and beyond the 140 kb MEIS1 genomic locus through a combination of in silico conservation analysis, DNase I hypersensitivity analysis and histone H3 acetylation profiling in several human leukemic cell lines. To date, we have identified more than eight critical cis regulatory regions in the human MEIS1 genomic locus. In the current study, we focused on the region between -500 bp to +20 bp of the annotated human MEIS1 transcriptional start site (AHTSS), the putative promoter of this gene. This region contains a CpG island which is highly conserved throughout evolution with more than 70% identity between human and zebrafish in the region between 267 bp to 403 bp upstream of AHTSS. This region also displays DNase I hypersensitivity, significant enrichment of histone H3 acetylation and DNA hypomethylation in MEIS1 expressing cell lines such as K562. To better define the regions necessary for MEIS1 promoter activity we tested a deletion series spanning the -500 bp to +20 bp relative to AHTSS region in the pGL3 luciferase reporter vector in K562 cells. Truncation of the region 305 bp to 268 bp resulted in a strong decrease (∼6-fold) in promoter activity. Within this 38 bp region we identified several predicted transcription factor binding sites, including sites for Serum response factor, RUNX1 and ETS family member. Mutations or deletions of the predicted ETS family member binding site resulted in a decrease of promoter activity to almost basal levels whereas mutations of the other predicted binding sites did not significantly alter promoter activity. We next tested the involvement of three ETS family members in MEIS1 regulation: ELF1, FLI1 and GABPa. Both ELF1 and GABPa, but not FLI1, bound to the predicted ETS family member binding site as assessed by electrophoretic mobility shift assay conducted with nuclear extracts from cells with or without detectable MEIS1 expression, K562 and HL60, respectively. However, chromatin immunoprecipitation assay revealed occupancy of ELF1, but not GABPa or FLI1, to the MEIS1 promoter only in MEIS1 expressing K562 cells. Moreover, siRNA knockdown of ELF1 in K562 cells was associated with decreased MEIS1 expression. Together, these findings implicate the ETS family member ELF1 as a key regulator of MEIS1 expression. The absence of MEIS1 expression in HL60 cells expressing ELF1 argues that additional regulators remain to be identified. Studies of additional candidate cis-regulatory regions in the MEIS1 locus which contribute to the overall chromatin status and the expression of this critical leukemogenic gene are now in progress. Disclosures:No relevant conflicts of interest to declare.

In Silico Detection of Sequence Variations Modifying Transcriptional Regulation

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

Richard Paul Bunge, M.D.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for ∼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems.