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。     

In silico identification of short nucleotide sequences associated with gene expression of pollen development in rice

Microarray analysis of tiny amounts of RNA extracted from plant section samples prepared by laser microdissection (LM) can provide high-quality information on gene expression in specified plant cells at various stages of development. Having joined the LM-microarray analysis project, we utilized such genome-wide gene expression data from developing rice pollen cells to identify candidates for cis-regulatory elements for specific gene expression in these cells. We first found a few clusters of gene expression patterns based on the data from LM-microarrays. On one gene cluster in which the members were specifically expressed at the bicellular and mature pollen mitotic stages, we identified gene cluster fingerprints (GCFs), each of which consists of a short nucleotide representing the gene cluster. We expected that these GCFs would contain cis-regulatory elements for stage- and tissue-specific gene expression, and we further identified groups of GCFs with common core sequences. Some criteria, such as frequency of occurrence in the gene cluster in contrast to the total tested gene set, flanking sequence preference and distribution of combined GCF sets in the gene regions, allowed us to limit candidates for cis-regulatory sequences for specific gene expression in rice pollen cells to at least 20 sets of combined GCFs. This approach should provide a general purpose algorithm for identifying short nucleotides associated with specific gene expression.     

Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence

Cis-regulatory sequences, such as enhancers and promoters, control development and physiology by regulating gene expression. Mutations that affect the function of these sequences contribute to phenotypic diversity within and between species. With many case studies implicating divergent cis-regulatory activity in phenotypic evolution, researchers have recently begun to elucidate the genetic and molecular mechanisms that are responsible for cis-regulatory divergence. Approaches include detailed functional analysis of individual cis-regulatory elements and comparing mechanisms of gene regulation among species using the latest genomic tools. Despite the limited number of mechanistic studies published to date, this work shows how cis-regulatory activity can diverge and how studies of cis-regulatory divergence can address long-standing questions about the genetic mechanisms of phenotypic evolution.     

Nomadic enhancers: tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species

cis-regulatory DNA sequences known as enhancers control gene expression in space and time. They are central to metazoan development and are often responsible for changes in gene regulation that contribute to phenotypic evolution. Here, we examine the sequence, function, and genomic location of enhancers controlling tissue- and cell-type specific expression of the yellow gene in six Drosophila species. yellow is required for the production of dark pigment, and its expression has evolved largely in concert with divergent pigment patterns. Using Drosophila melanogaster as a transgenic host, we examined the expression of reporter genes in which either 5' intergenic or intronic sequences of yellow from each species controlled the expression of Green Fluorescent Protein. Surprisingly, we found that sequences controlling expression in the wing veins, as well as sequences controlling expression in epidermal cells of the abdomen, thorax, and wing, were located in different genomic regions in different species. By contrast, sequences controlling expression in bristle-associated cells were located in the intron of all species. Differences in the precise pattern of spatial expression within the developing epidermis of D. melanogaster transformants usually correlated with adult pigmentation in the species from which the cis-regulatory sequences were derived, which is consistent with cis-regulatory evolution affecting yellow expression playing a central role in Drosophila pigmentation divergence. Sequence comparisons among species favored a model in which sequential nucleotide substitutions were responsible for the observed changes in cis-regulatory architecture. Taken together, these data demonstrate frequent changes in yellow cis-regulatory architecture among Drosophila species. Similar analyses of other genes, combining in vivo functional tests of enhancer activity with in silico comparative genomics, are needed to determine whether the pattern of regulatory evolution we observed for yellow is characteristic of genes with rapidly evolving expression patterns.     

Distinct functional constraints partition sequence conservation in a cis-regulatory element

Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter.     

Progress and challenges in studies of the evolution of development

Plant evolutionary developmental genetics (EDG) has made considerable progress over the last decade. This is in part due to the accumulation of large amounts of sequence data that have provided robust organismal phylogenies and, increasingly, broad assessments of molecular evolution. Attempts to use primary sequence data to identify genes that have changed function in evolutionary time have not been as successful as initially hoped. The coding sequences of most genes, which are more amenable to statistical analysis than are regulatory sequences, are generally under purifying selection, as would be expected if much evolutionary change is the result of changes in cis-regulatory sequences. Sequence-based analysis of the regulatory sequences themselves remains difficult. Comparative studies of gene expression have been useful to identify genes whose developmental role may have changed in evolutionary time and will be critical to the future development of EDG. Such studies can be used to test hypotheses of gene function. Transformation experiments are often illuminating, but can be hard to interpret, particularly if genes from multiple species are all placed into a single heterologous system such as Arabidopsis. The ideal experiment would be a gene swap or promoter swap between two species, but this awaits development of good transformation systems. The immediate need for EDG is studies of gene expression on a massive scale, far broader than any studies undertaken to date.     

Surveying phylogenetic footprints in large gene clusters: applications to Hox cluster duplications

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory regions. Since these elements are subject to stabilizing selection they evolve much more slowly than adjacent non-functional DNA. These so-called phylogenetic footprints can be detected by comparison of the sequences surrounding orthologous genes in different species. Therefore the loss of phylogenetic footprints as well as the acquisition of conserved non-coding sequences in some lineages, but not in others, can provide evidence for the evolutionary modification of cis-regulatory elements. We introduce here a statistical model of footprint evolution that allows us to estimate the loss of sequence conservation that can be attributed to gene loss and other structural reasons. This approach to studying the pattern of cis-regulatory element evolution, however, requires the comparison of relatively long sequences from many species. We have therefore developed an efficient software tool for the identification of corresponding footprints in long sequences from multiple species. We apply this novel method to the published sequences of HoxA clusters of shark, human, and the duplicated zebrafish and Takifugu clusters as well as the published HoxB cluster sequences. We find that there is a massive loss of sequence conservation in the intergenic region of the HoxA clusters, consistent with the finding in [Chiu et al., PNAS 99 (2002) 5492]. The loss of conservation after cluster duplication is more extensive than expected from structural reasons. This suggests that binding site turnover and/or adaptive modification may also contribute to the loss of sequence conservation.     

Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.