The structure and evolution of cis-regulatory regions: the shavenbaby story

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.     

Exclusive developmental functions of gatae cis-regulatory modules in the Strongylocentrorus purpuratus embryo

The gatae gene of Strongylocentrotus purpuratus is orthologous to vertebrate gata-4,5,6 genes. This gene is expressed in the endomesoderm in the blastula and later the gut of the embryo, and is required for normal development. A gatae BAC containing a GFP reporter knocked into exon one of the gene was able to reproduce all aspects of endogenous gatae expression in the embryo. To identify putative gatae cis-regulatory modules we carried out an interspecific sequence conservation analysis with respect to a Lytechinus variegatus gatae BAC, which revealed 25 conserved non-coding sequence patches. These were individually tested in gene transfer experiments, and two modules capable of driving localized reporter expression in the embryo were identified. Module 10 produces early expression in mesoderm and endoderm cells up to the early gastrula stage, while module 24 generates late endodermal expression at gastrula and pluteus stages. Module 10 was then deleted from the gatae BAC by reciprocal recombination, resulting in total loss of reporter expression in the time frame in which it is normally active. Similar deletion of module 24 led to ubiquitous GFP expression in the gastrula and pluteus. These results show that Module 10 is uniquely necessary and sufficient to account for the early phase of gatae expression during endomesoderm specification. In addition, they imply a functional cis-regulatory module exclusion, whereby only a single module can associate with the basal promoter and drive gene expression at any given time.     

RNA-ID,a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.     

Effects of gene regulatory reprogramming on gene expression in human and mouse developing hearts

Lineage-specific regulatory elements underlie adaptation of species and play a role in disease susceptibility. We compared functionally conserved and lineage-specific enhancers by cross-mapping 5042 human and 6564 mouse heart enhancers. Of these, 79 per cent are lineage-specific, lacking a functional orthologue. Heart enhancers tend to cluster and, commonly, there are multiple heart enhancers in a heart locus providing a regulatory stability to the locus. We observed little cross-clustering, however, between lineage-specific and functionally conserved heart enhancers suggesting regulatory function acquisition and development in loci previously lacking heart activity. We also identified 862 human-specific heart enhancers: 417 featuring sequence conservation with mouse (class II) and 445 with neither sequence nor function conservation (class III). Ninety-eight per cent of class III enhancers were deleted from the mouse genome, and we estimated a similar-sized enhancer gain in the human lineage. Human-specific enhancers display no detectable decrease in the negative selection pressure and are strongly associated with genes partaking in the heart regulatory programmes. The loss of a heart enhancer could be compensated by activity of a redundant heart enhancer; however, we observed redundancy in only 15 per cent of class II and III enhancer loci indicating a large-scale reprogramming of the heart regulatory programme in mammals.     

Repeated phenotypic changes highlight molecular targets of convergent evolution

How predictable is evolution at the molecular level? An example of repeated evolution in rice and Brassica illustrates how selection might preferentially target certain genes and mutations.     

水稻OsWRKY89启动子紫外线-B反应元件区的鉴定

植物需要利用太阳光能进行光合作用,因而不可避免地受到紫外线-B(UV-B) 辐射的影响．为了鉴定水稻WRKY转 录因子OsWRKY89基因启动子中的UV-B反应相关元件,分析了转启动子不同缺失片段与gus融合基因的水稻幼苗,发现在该启动子中存在UV-B反应元件,位于基因翻译起始位点上游-1 213～-1 188之间的25 bp区域,碱基序列为AAGATCTACCATTGCTCTATAGCTT．结合OsWRKY89和UV-B诱导上调表达基因启动子序列分析发现,该元件区在水稻UV-B反应基因启动子上具有高度的保守性,而且与已知保守的光反应元件位置邻近,表明该区域在水稻UV-B反应的转录调控中可能具有重要功能．     

Global spread and genetic variants of the two CYP9M10 haplotype forms associated with insecticide resistance in Culex quinquefasciatus Say

Insecticide resistance develops as a genetic factor (allele) conferring lower susceptibility to insecticides proliferates within a target insect population under strong positive selection. Intriguingly, a resistance allele pre-existing in a population often bears a series of further adaptive allelic variants through new mutations. This phenomenon occasionally results in replacement of the predominating resistance allele by fitter new derivatives, and consequently, development of greater resistance at the population level. The overexpression of the cytochrome P450 gene CYP9M10 is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. Previously, we have found two genealogically related overexpressing CYP9M10 haplotypes, which differ in gene copy number (duplicated and non-duplicated). The duplicated haplotype was derived from the non-duplicated overproducer probably recently. In the present study, we investigated allelic series of CYP9M10 involved in three C. quinquefasciatus laboratory colonies recently collected from three different localities. Duplicated and non-duplicated overproducing haplotypes coexisted in African and Asian colonies indicating a global distribution of both haplotype lineages. The duplicated haplotypes both in the Asian and African colonies were associated with higher expression levels and stronger resistance than non-duplicated overproducing haplotypes. There were slight variation in expression level among the non-duplicated overproducing haplotypes. The nucleotide sequences in coding and upstream regions among members of this group also showed a little diversity. Non-duplicated overproducing haplotypes with relatively higher expression were genealogically closer to the duplicated haplotypes than the other non-duplicated overproducing haplotypes, suggesting multiple cis-acting mutations before duplication.