果蝇中顺式调控序列的进化

顺式调控序列(cis-regularory sequences)是与基因表达调控相关、能够被调控因子特异性识别和结合的非编码DNA序列。顺式调控序列可以通过增减所含转录因子结合位点的数目,重构转录调控网络,以精准调控基因的时空表达模式,从而调控动物的生理和形态表型。顺式调控假说认为顺式调控序列进化是自然界丰富的动物表型进化的主要遗传机制。本文首先简述了顺式调控序列的结构和功能,然后重点讨论了近年来顺式调控序列进化调控果蝇表型进化如刚毛表型进化、色素沉积表型进化和胚胎发育方面的研究进展,阐释了顺式调控序列进化是动物表型进化的主要遗传机制之一。最后本文展望了顺式调控序列未来研究方向,例如应用功能基因组研究、开展ENCODE计划等,为进化发育生物学中顺式调控序列的研究提供了参考。     

Evolution of lineage-specific functions in ancient cis-regulatory modules

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.     

Evolution of cis-regulatory sequence and function in Diptera

Cis-regulatory sequences direct patterns of gene expression essential for development and physiology. Evolutionary changes in these sequences contribute to phenotypic divergence. Despite their importance, cis-regulatory regions remain one of the most enigmatic features of the genome. Patterns of sequence evolution can be used to identify cis-regulatory elements, but the power of this approach depends upon the relationship between sequence and function. Comparative studies of gene regulation among Diptera reveal that divergent sequences can underlie conserved expression, and that expression differences can evolve despite largely similar sequences. This complex structure-function relationship is the primary impediment for computational identification and interpretation of cis-regulatory sequences. Biochemical characterization and in vivo assays of cis-regulatory sequences on a genomic-scale will relieve this barrier.     

Evolution of cis-regulatory elements in duplicated genes of yeast

An increasing number of studies report that functional divergence in duplicated genes is accompanied by gene expression changes, although the evolutionary mechanism behind this process remains unclear. Our genomic analysis on the yeast Saccharomyces cerevisiae shows that the number of shared regulatory motifs in the duplicates decreases with evolutionary time, whereas the total number of regulatory motifs remains unchanged. Moreover, genes with numerous paralogs in the yeast genome do not have especially low number of regulatory motifs. These findings indicate that degenerative complementation is not the sole mechanism behind expression divergence in yeast. Moreover, we found some evidence for the action of positive selection on cis-regulatory motifs after gene duplication. These results suggest that the evolution of functional novelty has a substantial role in yeast duplicate gene evolution.     

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.     

Gene expression in time and space: additive vs hierarchical organization of cis-regulatory regions

In higher eukaryotes, individual genes are often intermingled with other genes and spread out across tens to hundreds of kilobases, even though only small portions of their sequence are devoted to protein coding. Yet, in this seemingly extended and tangled mess, the cell is able to precisely regulate gene expression in both time and space. Over the past few decades, numerous elements, like enhancers, silencers and insulators have been found that shed some light on how the precise control of gene expression is achieved. Through these discoveries, an additive model of gene expression was envisioned, where the addition of the patterning details imparted by regulatory elements would create the final pattern of gene expression. Although many genes can be described using this model, recent work in the Drosophila bithorax complex suggests that this model may be somewhat simplistic and, in fact, regulatory elements sometimes seem to communicate with each other to form a functional hierarchy that is far from additive.     

Molecular evolution of duplicated amylase gene regions in Drosophila melanogaster: evidence of positive selection in the coding regions and selective constraints in the cis-regulatory regions

In this study, we randomly sampled Drosophila melanogaster from Japanese and Kenyan natural populations. We sequenced duplicated (proximal and distal) Amy gene regions to test whether the patterns of polymorphism were consistent with neutral molecular evolution. F(st) between the two geographically distant populations, estimated from Amy gene regions, was 0.084, smaller than reported values for other loci, comparing African and Asian populations. Furthermore, little genetic differentiation was found at a microsatellite locus (DROYANETSB) in these samples (G'st = -0.018). The results of several tests (Tajima's, Fu and Li's, and Wall's tests) were not significantly different from neutrality. However, a significantly higher level of fixed replacement substitutions was detected by a modified McDonald and Kreitman test for both populations. This indicates that positive selection occurred during or immediately after the speciation of D. melanogaster. Sliding-window analysis showed that the proximal region 1, a part of the proximal 5' flanking region, was conserved between D. melanogaster and its sibling species, D. simulans. An HKA test was significant when the proximal region 1 was compared with the 5' flanking region of Alcohol dehydrogenase (Adh), indicating a severe selective constraint on the Amy proximal region 1. These results suggest that natural selection has played an important role in the molecular evolution of Amy gene regions in D. melanogaster.     

Evolution of wing pigmentation in Drosophila: Diversity,physiological regulation,and cis-regulatory evolution

Fruit flies (Drosophila and its close relatives, or “drosophilids”) are a group that includes an important model organism, Drosophila melanogaster, and also very diverse species distributed worldwide. Many of these species have black or brown pigmentation patterns on their wings, and have been used as material for evo-devo research. Pigmentation patterns are thought to have evolved rapidly compared with body plans or body shapes; hence they are advantageous model systems for studying evolutionary gains of traits and parallel evolution. Various groups of drosophilids, including genus Idiomyia (Hawaiian Drosophila), have a variety of pigmentations, ranging from simple black pigmentations around crossveins to a single antero-distal spot and a more complex mottled pattern. Pigmentation patterns are sometimes obviously used for sexual displays; however, in some cases they may have other functions. The process of wing formation in Drosophila, the general mechanism of pigmentation formation, and the transport of substances necessary for pigmentation, including melanin precursors, through wing veins are summarized here. Lastly, the evolution of the expression of genes regulating pigmentation patterns, the role of cis-regulatory regions, and the conditions required for the evolutionary emergence of pigmentation patterns are discussed. Future prospects for research on the evolution of wing pigmentation pattern formation in drosophilids are presented, particularly from the point of view of how they compare with other studies of the evolution of new traits.     

Evolution of the noncoding regions inDrosophila mitochondrial DNA: Two intergenic regions

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to theDrosophila immigrans species group and compared to the corresponding segments ofDrosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between thetRNA ^gln and thetRNA ^ile genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between thetRNA ^f-met and thetRNA ^ile genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in thenasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.     

Positive and negative cis-regulatory regions in the soybean glycinin promoter identified by quantitative transient gene expression

Summary The 5 and 3 flanking regions of the soybean glycinin gene, Gy1, responsible for expression in seeds, were analyzed by quantitative transient expression assay. The construct containing the -glucuronidase (uidA) reporter gene under the control of the 1.12 kb Gy1 promoter and 0.74 kb Gy1 terminator was introduced into immature soybean seeds and leaves by particle bombardment. To normalize the variability of introduction efficiency, a second reporter gene, firefly luciferase, was cobombarded as an internal standard, and relative activities (GUS/luciferase) were measured. There was a seed-specific -glucuronidase (GUS) expression, as observed by X-Gluc staining. Compared with the nopaline synthase gene (nos) terminator, the Gy1 terminator enhanced the level of expression in immature seeds, indicating that the terminator region of the glycinin gene is involved in the activation of the gene expression in these seeds. To identify cis-regulatory elements in the glycinin gene upstream sequence, deleted derivatives of the promoter were fused to the luciferase reporter gene. The expression could be measured with a higher accuracy, and constructs were introduced with the internal reporter uidA gene into immature seeds. The results suggest the presence of a positive regulatory element in the –620 to ––380 region of the Gy1 promoter. A deletion which eliminates the legumin box with its RY element led to increased relative activity, suggesting that this box is negatively regulating expression of the seed storage protein gene. Analysis of mutant promoters also suggest that the RY element involves negative regulation in seeds. This quantitative transient expression assay using particle bombardment provides a reliable system for the study of seed-specific gene expression in soybeans.Abbreviations GUS -glucuronidase - Gy1 glycinin AlaB2 gene - CaMV cauliflower mosaic virus - nos nopaline synthase gene - uidA -glucuronidase gene - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Identification of novel cis-regulatory regions from the Notch receptor genes lin-12 and glp-1 of Caenorhabditis elegans

Notch signaling regulates various cellular processes such as growth, proliferation and differentiation, and plays a key role in tissue patterning during animal development. In humans, defects in Notch signaling have been implicated in cancer, stroke, neurodegeneration, as well as learning and memory deficits. The genome of the nematode Caenorhabditis elegans encodes two members of the Notch transmembrane receptor family, LIN-12 and GLP-1, which have both unique and shared developmental functions. LIN-12 affects diverse cell fate specification events at certain embryonic and larval stages, including the ABplp lineage (a neuronal precursor), intestinal primordium, gonadal anchor cell and secondary vulval precursor cells. In addition to developmental functions, it also operates in the adult nervous system to control locomotion, memory and chemosensory response. Although lin-12 expression was subjected to intense analysis, it was almost not demonstrable in neurons; occasional lin-12 expression was detected only in the two RIG interneurons of young larvae. Here we identify two cis-regulatory regions from lin-12, both of them are specified by the presence of a conserved EXD/HOX composite binding site. One of these regions is located in the first intron and required for driving transgene expression in vulval precursor cell lineages and specific gonadal cells. The other region is located in the second intron and can confer neuronal expression for lin-12 throughout life. The latter regulatory element is highly conserved in the paralogous glp-1 genomic environment, suggesting redundant developmental and physiological roles for the two Notch paralogs in the C. elegans nervous system.     

Characterization of cis-regulatory regions responsible for developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana

The Arabidopsis GA1 gene encodes copalyl diphosphate synthase, which catalyzes the first committed step in the gibberellin biosynthetic pathway. Previous studies indicated that the expression pattern of the GA1 gene is tissue-specific and cell-type-specific during development. Here we showed that expression of GA1 cDNA driven by the 2.4 kb 5-upstream sequence plus the GA1 genomic coding region into the third exon was able to rescue the ga1-3 mutant phenotype. To understand the mechanism controlling GA1 gene expression, cis-regulatory regions in the GA1 promoter were identified by promoter deletion analysis with the GA1--glucuronidase (GUS) gene fusion system. The second intron and the region from –1391 to –997, with respect to the translation initiation site, positively regulate overall GA1-GUS expression level in all tissues examined. Several additional regulatory regions are involved in GA1-GUS expression in all the stages except in seeds: two positive regulatory regions in the first intron and the sequence between –425 and –207, and a negative regulatory region between –1848 and –1391. We also found that the region between –997 and –796 is essential for a high level of GA1 expression in developing seeds.