Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.     

Synthetic Promoters: Designing the <Emphasis Type="Italic">cis</Emphasis> Regulatory Modules for Controlled Gene Expression

Designing the expression cassettes with desired properties remains the most important consideration of gene engineering technology. One of the challenges for predictive gene expression is the modeling of synthetic gene switches to regulate one or more target genes which would directly respond to specific chemical, environmental, and physiological stimuli. Assessment of natural promoter, high-throughput sequencing, and modern biotech inventory aided in deciphering the structure of cis elements and molding the native cis elements into desired synthetic promoter. Synthetic promoters which are molded by rearrangement of cis motifs can greatly benefit plant biotechnology applications. This review gives a glimpse of the manual in vivo gene regulation through synthetic promoters. It summarizes the integrative design strategy of synthetic promoters and enumerates five approaches for constructing synthetic promoters. Insights into the pattern of cis regulatory elements in the pursuit of desirable “gene switches” to date has also been reevaluated. Joint strategies of bioinformatics modeling and randomized biochemical synthesis are addressed in an effort to construct synthetic promoters for intricate gene regulation.     

细菌转录起始调控机制*

原核生物同一种群的每个细胞都是和外界环境直接接触的,它们主要通过开启或关闭某些基因的表达来适应环境条件。所以,环境因子往往是调控的效应因子,必须严格调控转录来确保细胞对环境改变做出有效且充分的反应。原核生物基因的表达受多种因素的调控,而对于大多数细菌来说,调控基因表达的关键步骤是启动子识别和RNA聚合酶启动转录。在细菌的细胞中,可以通过调节RNA聚合酶的活性以及改变RNA聚合酶对启动子的结合来优化基因的转录过程以适应不同环境变化。总结了目前已发现的参与细菌细胞转录调节的各类因子,从这些因子对启动子的作用、RNA聚合酶的作用以及两者的相互作用等方面阐述它们调控基因表达的分子机制。总结多种基因调控的作用,加深对转录起始过程的认识,希望能对未来调控转录起始过程来实现目标基因的高效表达和不利基因的抑制表达提供思路,为以后的工业菌株改造提供依据。     

Incorporating regulatory interactions into gene-set analyses for GWAS data: A controlled analysis with the MAGMA tool

To date, genome-wide association studies have identified thousands of statistically-significant associations between genetic variants, and phenotypes related to a myriad of traits and diseases. A key goal for human-genetics research is to translate these associations into functional mechanisms. Popular gene-set analysis tools, like MAGMA, map variants to genes they might affect, and then integrate genome-wide association study data (that is, variant-level associations for a phenotype) to score genes for association with a phenotype. Gene scores are subsequently used in competitive gene-set analyses to identify biological processes that are enriched for phenotype association. By default, variants are mapped to genes in their proximity. However, many variants that affect phenotypes are thought to act at regulatory elements, which can be hundreds of kilobases away from their target genes. Thus, we explored the idea of augmenting a proximity-based mapping scheme with publicly-available datasets of regulatory interactions. We used MAGMA to analyze genome-wide association study data for ten different phenotypes, and evaluated the effects of augmentation by comparing numbers, and identities, of genes and gene sets detected as statistically significant between mappings. We detected several pitfalls and confounders of such “augmented analyses”, and introduced ways to control for them. Using these controls, we demonstrated that augmentation with datasets of regulatory interactions only occasionally strengthened the enrichment for phenotype association amongst (biologically-relevant) gene sets for different phenotypes. Still, in such cases, genes and regulatory elements responsible for the improvement could be pinpointed. For instance, using brain regulatory-interactions for augmentation, we were able to implicate two acetylcholine receptor subunits involved in post-synaptic chemical transmission, namely CHRNB2 and CHRNE, in schizophrenia. Collectively, our study presents a critical approach for integrating regulatory interactions into gene-set analyses for genome-wide association study data, by introducing various controls to distinguish genuine results from spurious discoveries.     

Use of transposon promoter-probe vectors in the metabolic engineering of the obligate methanotroph Methylomonas sp. strain 16a for enhanced C40 carotenoid synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.     

The genetic basis of sexual dimorphism in birds

The genetic basis of sexual dimorphisms is an intriguing problem of evolutionary genetics because dimorphic traits are limited to one sex. Such traits can arise genetically in two ways. First, the alleles that cause dimorphisms could be limited in expression to only one sex at their first appearance. Alternatively, dimorphism alleles could initially be expressed in both sexes, but subsequently be repressed or promoted in only one sex by the evolution of modifier genes or regulatory elements. We investigated these alternatives by looking for the expression of sexually dimorphic traits in female hybrids between bird species whose males show different types of ornaments. If modifier alleles or regulatory elements involved in sex-limited traits are not completely dominant, the modification should break down in female hybrids, which might then show dimorphic traits resembling those seen in males. Of 13 interspecific hybridizations examined, we found not a single instance of the expression of male-limited ornaments in female hybrids. This suggests that male ornaments were sex limited from the outset or that those traits became sex limited through the evolution of dominant modifiers -- possibly cis-dominant regulatory elements. Observing hybrid phenotypes is a useful approach to studying the genetics and evolution of dimorphic traits.