A Far-Upstream Sequence of the Wheat Histone H3 Promoter Functions Differently in Rice and Tobacco Cultured Cells

The cis-regulatory function of a far-upstream sequence (–1,711to –186) of the promoter of the wheat gene for histoneH3 (TH012) was analyzed in cultured rice and tobacco cells ina transient expression system with the gene for rß-D-glucuronidaseas a reporter gene. The far-upstream sequence was necessaryfor full activity of the H3 promoter in rice cells but did notenhance the activity of the proximal promoter in tobacco cells.Dissection analysis of the far-upstream sequence revealed theexistence of several positive and negative cis-acting sequencesin this region, some of which functioned differently in riceand tobacco cells. In gain-of-function experiments with ricecells, the sequence from –848 to –704, containingthe CCAAT and octamer (CaCGGATC) motifs, functioned in an orientation-independentmanner, whereas the sequence from –703 to –486 functionedin an orientation-dependent manner. By contrast, both sequencesexhibited an orientation-dependent cis-function in tobacco cells.These findings suggest that some cis-regulatory sequences inthe far-upstream region of the H3 promoter function differentlyin rice and tobacco cells. (Received May 10, 1995; Accepted August 8, 1995)     

Analysis of the genetic loci of pigment pattern evolution in vertebrates

Vertebrate pigmentation patterns are amongst the best characterised model systems for studying the genetic basis of adaptive evolution. The wealth of available data on the genetic basis for pigmentation evolution allows for analysis of trends and quantitative testing of evolutionary hypotheses. We employed Gephebase, a database of genetic variants associated with natural and domesticated trait variation, to examine trends in how cis-regulatory and coding mutations contribute to vertebrate pigmentation phenotypes, as well as factors that favour one mutation type over the other. We found that studies with lower ascertainment bias identified higher proportions of cis-regulatory mutations, and that cis-regulatory mutations were more common amongst animals harbouring a higher number of pigment cell classes. We classified pigmentation traits firstly according to their physiological basis and secondly according to whether they affect colour or pattern, and identified that carotenoid-based pigmentation and variation in pattern boundaries are preferentially associated with cis-regulatory change. We also classified genes according to their developmental, cellular, and molecular functions. We found a greater proportion of cis-regulatory mutations in genes implicated in upstream developmental processes compared to those involved in downstream cellular functions, and that ligands were associated with a higher proportion of cis-regulatory mutations than their respective receptors. Based on these trends, we discuss future directions for research in vertebrate pigmentation evolution.     

Intra-Specific Regulatory Variation in Drosophila pseudoobscura

It is generally accepted that gene regulation serves an important role in determining the phenotype. To shed light on the evolutionary forces operating on gene regulation, previous studies mainly focused on the expression differences between species and their inter-specific hybrids. Here, we use RNA-Seq to study the intra-specific distribution of cis- and trans-regulatory variation in Drosophila pseudoobscura. Consistent with previous results, we find almost twice as many genes (26%) with significant trans-effects than genes with significant cis-effects (18%). While this result supports the previous suggestion of a larger mutational target of trans-effects, we also show that trans-effects may be subjected to purifying selection. Our results underline the importance of intra-specific analyses for the understanding of the evolution of gene expression.     

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.     

A Robust Approach to Identifying Tissue-Specific Gene Expression Regulatory Variants Using Personalized Human Induced Pluripotent Stem Cells

Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS) cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.     

Internet Resources for Gene Expression Analysis in Arabidopsis thaliana

The number of online databases and web-tools for gene expression analysis in Arabidopsis thaliana has increased tremendously during the last years. These resources permit the database-assisted identification of putative cis-regulatory DNA sequences, their binding proteins, and the determination of common cis-regulatory motifs in coregulated genes. DNA binding proteins may be predicted by the type of cis-regulatory motif. Further questions of combinatorial control based on the interaction of DNA binding proteins and the colocalization of cis-regulatory motifs can be addressed. The database-assisted spatial and temporal expression analysis of DNA binding proteins and their target genes may help to further refine experimental approaches. Signal transduction pathways upstream of regulated genes are not yet fully accessible in databases mainly because they need to be manually annotated. This review focuses on the use of the AthaMap and PathoPlant^® databases for gene expression regulation analysis and discusses similar and complementary online databases and web-tools. Online databases are helpful for the development of working hypothesis and for designing subsequent experiments.