Evolution Of Cis-regulatory Elements Research Articles

97 Dissection of Evolution of Cis-Regulatory Elements and Its Application on Genetic Control of Complex Traits in Farm Animals

Abstract Functional Annotation of Animal Genomes (FAANG) has made significant progress on the improvement of the annotation of the functional elements, particularly non-coding regions in farm animal genomes. Different epigenomes including ChIP-seq for 4 histone marks, ATAC-seq, CTCF-seq, RNA-seq, and Hi-C across different tissues in different animal species were generated. Thousands of cis-regulatory elements including promoters and enhancers across different tissues in different species were identified and annotated. A comprehensive characterization and comparison of cis-regulatory elements across tissues and species can help us understand the genetic and evolutionary basis of complex phenotypes. Yet, attempts to dissect the evolution of regulatory elements have generally been small, both regarding the number of tissues and species compared. We demonstrate that genomic variants associated with complex traits and adaptive evolution in farm animals are significantly enriched in active promoters and enhancers. Further analysis suggests that the regulators were significantly enriched in evolutionary breakpoint regions (EBRs) in a tissue-specific and regulator-specific manner. Furthermore, we reveal distinct tissue-specific regulatory elements play important roles in domestication processes. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating genome-wide association studies on complex traits, we demonstrate that comparative epigenomic analysis enhances our interpretations of complex traits variations in farm animals and provides novel insights on the role of regulatory elements on the functional conservation across species.

Comparative phylogenomic analysis of 5’cis-regulatory elements (CREs) of miR160 gene family in diploid and allopolyploid cotton (Gossypium)

Cotton fiber development is a complex process regulated by numerous microRNAs (miRNAs), including miR160 that regulates auxin signaling in a grwoing cell. However, the evolution of cis-regulatory elements (CREs) in the 5’upstream regulatory regions of miR160 genes during cotton allopolyploidy is least known. Therefore, 1.5 kb upstream DNA sequences (−1 to −1500 bp) of miR160 genes from progenitor diploid A2 (G. arboreum), D5 (G. raimondii) and descendant allopolyploid AD1 (G. hirsutum) and AD2 (G. barbadense) cotton species were analyzed to visualize the evolutionary patterns preserved within the promoter regions of these homologous sequences. Several light-responsive, developmental-responsive, organ-specific, stress-responsive (biotic and abiotic), and hormone-responsive CREs were identified with diverse frequencies and wide distribution patterns. The structural alterations in the promoter regions were more prominent than the evolutionary changes acquired by miR160 genes, suggesting that miR160 gene sequences have been conserved throughout evolution, and the observed expression variations across cotton species are primarily due to the structural differences of their regulatory regions. The abiotic stress-responsive and hormone-responsive CREs exhibited prominent clustering at both proximal and distal ends, displaying varying frequencies. Thus, the origin of these CREs can be attributed to the genomic hybridization of diploid species that had led to the allopolyploid formation, or to the natural speciation/diversification of an allopolyploid event. Also, there is a high correlation between CREs-binding transcription factors and miR160 gene expression, suggesting a regulatory mechanism for spatiotemporal expression profiles of miR160 genes during cotton fiber development.

Roles of transposable elements in the regulation of mammalian transcription.

Transposable elements (TEs) comprise about half of the mammalian genome. TEs often contain sequences capable of recruiting the host transcription machinery, which they use to express their own products and promote transposition. However, the regulatory sequences carried by TEs may affect host transcription long after the TEs have lost the ability to transpose. Recent advances in genome analysis and engineering have facilitated systematic interrogation of the regulatory activities of TEs. In this Review, we discuss diverse mechanisms by which TEs contribute to transcription regulation. Notably, TEs can donate enhancer and promoter sequences that influence the expression of host genes, modify 3D chromatin architecture and give rise to novel regulatory genes, including non-coding RNAs and transcription factors. We discuss how TEs spur regulatory evolution and facilitate the emergence of genetic novelties in mammalian physiology and development. By virtue of their repetitive and interspersed nature, TEs offer unique opportunities to dissect the effects of mutation and genomic context on the function and evolution of cis-regulatory elements. We argue that TE-centric studies hold the key to unlocking general principles of transcription regulation and evolution.

Disparate Dynamics of Gene Body and cis-Regulatory Element Evolution Illustrated for the Senescence-Associated Cysteine Protease Gene SAG12 of Plants.

Gene regulation networks precisely orchestrate the expression of genes that are closely associated with defined physiological and developmental processes such as leaf senescence in plants. The Arabidopsis thaliana senescence-associated gene 12 (AtSAG12) encodes a cysteine protease that is (i) involved in the degradation of chloroplast proteins and (ii) almost exclusively expressed during senescence. Transcription factors, such as WRKY53 and WRKY45, bind to W-boxes in the promoter region of AtSAG12 and play key roles in its activation. Other transcription factors, such as bZIPs, might have accessory functions in their gene regulation, as several A-boxes have been identified and appear to be highly overrepresented in the promoter region compared to the whole genome distribution but are not localized within the regulatory regions driving senescence-associated expression. To address whether these two regulatory elements exhibiting these different properties are conserved in other closely related species, we constructed phylogenetic trees of the coding sequences of orthologs of AtSAG12 and screened their respective 2000 bp promoter regions for the presence of conserved cis-regulatory elements, such as bZIP and WRKY binding sites. Interestingly, the functional relevant upstream located W-boxes were absent in plant species as closely related as Arabidopsis lyrata, whereas an A-box cluster appeared to be conserved in the Arabidopsis species but disappeared in Brassica napus. Several orthologs were present in other species, possibly because of local or whole genome duplication events, but with distinct cis-regulatory sites in different locations. However, at least one gene copy in each family analyzed carried one W-box and one A-box in its promoter. These gene differences in SAG12 orthologs are discussed in the framework of cis- and trans-regulatory factors, of promoter and gene evolution, of genetic variation, and of the enhancement of the adaptability of plants to changing environmental conditions.

Impact of Genetic Variation in Gene Regulatory Sequences: A Population Genomics Perspective.

The unprecedented rise of high-throughput sequencing and assay technologies has provided a detailed insight into the non-coding sequences and their potential role as gene expression regulators. These regulatory non-coding sequences are also referred to as cis-regulatory elements (CREs). Genetic variants occurring within CREs have been shown to be associated with altered gene expression and phenotypic changes. Such variants are known to occur spontaneously and ultimately get fixed, due to selection and genetic drift, in natural populations and, in some cases, pave the way for speciation. Hence, the study of genetic variation at CREs has improved our overall understanding of the processes of local adaptation and evolution. Recent advances in high-throughput sequencing and better annotations of CREs have enabled the evaluation of the impact of such variation on gene expression, phenotypic alteration and fitness. Here, we review recent research on the evolution of CREs and concentrate on studies that have investigated genetic variation occurring in these regulatory sequences within the context of population genetics.

Epigenomic landscape of enhancer elements during Hydra head organizer formation

BackgroundAxis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra.ResultsTo gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity.ConclusionsThe activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum.

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.

Cis-regulatory evolution integrated the Bric-à-brac transcription factors into a novel fruit fly gene regulatory network.

Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here, we show that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.

Evidence for the role of transposons in the recruitment of cis-regulatory motifs during the evolution of C4 photosynthesis.

BackgroundC4 photosynthesis evolved from C3 photosynthesis and has higher light, water, and nitrogen use efficiencies. Several C4 photosynthesis genes show cell-specific expression patterns, which are required for these high resource-use efficiencies. However, the mechanisms underlying the evolution of cis-regulatory elements that control these cell-specific expression patterns remain elusive.ResultsIn the present study, we tested the hypothesis that the cis-regulatory motifs related to C4 photosynthesis genes were recruited from non-photosynthetic genes and further examined potential mechanisms facilitating this recruitment. We examined 65 predicted bundle sheath cell-specific motifs, 17 experimentally validated cell-specific cis-regulatory elements, and 1,034 motifs derived from gene regulatory networks. Approximately 7, 5, and 1,000 of these three categories of motifs, respectively, were apparently recruited during the evolution of C4 photosynthesis. In addition, we checked 1) the distance between the acceptors and the donors of potentially recruited motifs in a chromosome, and 2) whether the potentially recruited motifs reside within the overlapping region of transposable elements and the promoter of donor genes. The results showed that 7, 4, and 658 of the potentially recruited motifs might have moved via the transposable elements. Furthermore, the potentially recruited motifs showed higher binding affinity to transcription factors compared to randomly generated sequences of the same length as the motifs.ConclusionsThis study provides molecular evidence supporting the hypothesis that transposon-driven recruitment of pre-existing cis-regulatory elements from non-photosynthetic genes into photosynthetic genes plays an important role during C4 evolution. The findings of the present study coincide with the observed repetitive emergence of C4 during evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2519-3) contains supplementary material, which is available to authorized users.

Large-scale gene expression study in the ophiuroid Amphiura filiformis provides insights into evolution of gene regulatory networks.

BackgroundThe evolutionary mechanisms involved in shaping complex gene regulatory networks (GRN) that encode for morphologically similar structures in distantly related animals remain elusive. In this context, echinoderm larval skeletons found in brittle stars and sea urchins provide an ideal system. Here, we characterize for the first time the development of the larval skeleton in the ophiuroid Amphiura filiformis and compare it systematically with its counterpart in sea urchin.ResultsWe show that ophiuroids and euechinoids, that split at least 480 Million years ago (Mya), have remarkable similarities in tempo and mode of skeletal development. Despite morphological and ontological similarities, our high-resolution study of the dynamics of genetic regulatory states in A. filiformis highlights numerous differences in the architecture of their underlying GRNs. Importantly, the A.filiformispplx, the closest gene to the sea urchin double negative gate (DNG) repressor pmar1, fails to drive the skeletogenic program in sea urchin, showing important evolutionary differences in protein function. hesC, the second repressor of the DNG, is co-expressed with most of the genes that are repressed in sea urchin, indicating the absence of direct repression of tbr, ets1/2, and delta in A. filiformis. Furthermore, the absence of expression in later stages of brittle star skeleton development of key regulatory genes, such as foxb and dri, shows significantly different regulatory states.ConclusionOur data fill up an important gap in the picture of larval mesoderm in echinoderms and allows us to explore the evolutionary implications relative to the recently established phylogeny of echinoderm classes. In light of recent studies on other echinoderms, our data highlight a high evolutionary plasticity of the same nodes throughout evolution of echinoderm skeletogenesis. Finally, gene duplication, protein function diversification, and cis-regulatory element evolution all contributed to shape the regulatory program for larval skeletogenesis in different branches of echinoderms.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-015-0039-x) contains supplementary material, which is available to authorized users.

Evolution of cis-regulatory elements and regulatory networks in duplicated genes of Arabidopsis thaliana

Plant genomes contain large numbers of duplicated genes that contribute to the evolution of new functions. Following duplication, genes can exhibit divergence in their coding sequence and their expression patterns. Changes in the cis-regulatory element landscape can result in changes in gene expression patterns. High-throughput methods developed recently can identify potential cis-regulatory elements on a genome-wide scale. Here, we use a recent comprehensive data set of DNase I sequencing-identified cis-regulatory binding sites (footprints) at single-base-pair resolution to compare binding sites and network connectivity in duplicated gene pairs in Arabidopsis (Arabidopsis thaliana). We found that duplicated gene pairs vary greatly in their cis-regulatory element architecture, resulting in changes in regulatory network connectivity. Whole-genome duplicates (WGDs) have approximately twice as many footprints in their promoters left by potential regulatory proteins than do tandem duplicates (TDs). The WGDs have a greater average number of footprint differences between paralogs than TDs. The footprints, in turn, result in more regulatory network connections between WGDs and other genes, forming denser, more complex regulatory networks than shown by TDs. When comparing regulatory connections between duplicates, WGDs had more pairs in which the two genes are either partially or fully diverged in their network connections, but fewer genes with no network connections than the TDs. There is evidence of younger TDs and WGDs having fewer unique connections compared with older duplicates. This study provides insights into cis-regulatory element evolution and network divergence in duplicated genes.

Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue.

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

Role of genes and their cis-regulatory elements during animal morphological evolution

Cis-regulatory hypothesis is one of the most important theories in evolutionary developmental biology (evo-devo), which claims that evolution of cis-regulatory elements (CREs) plays a key role during evolution of morphology. However, an increasing number of experimental results show that cis-regulatory hypothesis alone is not far enough to explain the complexity of evo-devo processes. Other modifications, including mutations of protein coding, gene and genome duplications, and flexibility of homeodomains and CREs, also cause the morphological changes in animals. In this review, we retrospect the recent results of evolution of CREs and genes associated with CREs and discuss new methods and trends for research in evo-devo.

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages.

Functional characterization of motif sequences under purifying selection.

Diverse life forms are driven by the evolution of gene regulatory programs including changes in regulator proteins and cis-regulatory elements. Alterations of cis-regulatory elements are likely to dominate the evolution of the gene regulatory networks, as they are subjected to smaller selective constraints compared with proteins and hence may evolve quickly to adapt the environment. Prior studies on cis-regulatory element evolution focus primarily on sequence substitutions of known transcription factor-binding motifs. However, evolutionary models for the dynamics of motif occurrence are relatively rare, and comprehensive characterization of the evolution of all possible motif sequences has not been pursued. In the present study, we propose an algorithm to estimate the strength of purifying selection of a motif sequence based on an evolutionary model capturing the birth and death of motif occurrences on promoters. We term this measure as the ‘evolutionary retention coefficient’, as it is related yet distinct from the canonical definition of selection coefficient in population genetics. Using this algorithm, we estimate and report the evolutionary retention coefficients of all possible 10-nucleotide sequences from the aligned promoter sequences of 27 748. orthologous gene families in 34 mammalian species. Intriguingly, the evolutionary retention coefficients of motifs are intimately associated with their functional relevance. Top-ranking motifs (sorted by evolutionary retention coefficients) are significantly enriched with transcription factor-binding sequences according to the curated knowledge from the TRANSFAC database and the ChIP-seq data generated from the ENCODE Consortium. Moreover, genes harbouring high-scoring motifs on their promoters retain significantly coherent expression profiles, and those genes are over-represented in the functional classes involved in gene regulation. The validation results reveal the dependencies between natural selection and functions of cis-regulatory elements and shed light on the evolution of gene regulatory networks.

Evolution of cis-regulatory elements in yeast de novo and duplicated new genes

BackgroundNew genes that originate from non-coding DNA rather than being duplicated from parent genes are called de novo genes. Their short evolution time and lack of parent genes provide a chance to study the evolution of cis-regulatory elements in the initial stage of gene emergence. Although a few reports have discussed cis-regulatory elements in new genes, knowledge of the characteristics of these elements in de novo genes is lacking. Here, we conducted a comprehensive investigation to depict the emergence and establishment of cis-regulatory elements in de novo yeast genes.ResultsIn a genome-wide investigation, we found that the number of transcription factor binding sites (TFBSs) in de novo genes of S. cerevisiae increased rapidly and quickly became comparable to the number of TFBSs in established genes. This phenomenon might have resulted from certain characteristics of de novo genes; namely, a relatively frequent gain of TFBSs, an unexpectedly high number of preexisting TFBSs, or lower selection pressure in the promoter regions of the de novo genes. Furthermore, we identified differences in the promoter architecture between de novo genes and duplicated new genes, suggesting that distinct regulatory strategies might be employed by genes of different origin. Finally, our functional analyses of the yeast de novo genes revealed that they might be related to reproduction.ConclusionsOur observations showed that de novo genes and duplicated new genes possess mutually distinct regulatory characteristics, implying that these two types of genes might have different roles in evolution.

Selection and constraint on regulatory elements in Drosophila simulans

We utilized available Drosophila simulans molecular population genomic data to characterize sequence polymorphism in noncoding regulatory regions and their corresponding transcribed target genes or interacting transcription factors. We highlight two properties of regulatory evolution. First, we find that sequence divergence between D. melanogaster and D. simulans is greater at regulatory sites than expected from levels of intraspecific polymorphism, suggestive of positive selection. Second, we compared variation in cis-regulatory regions to that of the transcription factors and transcribed target genes with which they are associated. We find a correlation in polymorphism between cis-regulatory regions and the transcription factors which they bind, but not between cis-regulatory regions and the physically neighboring target genes which they directly regulate. This result indicates that the evolution of cis-regulatory elements is constrained by the interactions among transcription factors and regulatory regions. More broadly, these findings support the general importance of noncoding regulatory DNA in evolution.

Evolution of vertebrate appendicular structures: Insight from genetic and palaeontological data

The new body of evidence from fossils and comparative-developmental analysis of subset of appendicular patterning genes has revealed that limb elements seen in tetrapods are assembled in fish fin over evolutionary time. However, despite of deep homology in basic structure and underlying developmental system, there remains a large morphological gap between distal elements of tetrapod limb and distal fin skeleton of tetrapodomorph fish. Understanding the genetic basis of major transformations in distal-limb morphology is the next challenge for evolutionary developmental biologists. Here by integrating data from fossils, comparative-developmental and genetic studies, models are proposed describing the evolution of cis-regulatory elements as a basis for diversification of appendicular architecture. Instead of emphasizing the subset of developmental genes, for instance Hoxd genes, the focus here is on the significance of elucidating cis-regulatory elements for multiple other key molecular players of limb/fin development and genetic/molecular interactions among them, for a better understanding of the developmental and genetic basis of limb evolution.

ER regulates an evolutionarily conserved apoptosis pathway

Estrogen receptor (ER) is regarded as a major causal factor in breast cancer and FoxA1, a winged-helix transcription factor belonging to the forkhead family, has been found to function as a pioneer factor in the recruitment of ER to several cis-regulatory elements in the genome. High throughput chromatin immunoprecipitation analyzed by hybridization to microarrays (ChIP-chip) can reveal ER and FoxA1 binding sites occupied by transcription factors. However, these results are blind to lineage-specific cis-regulatory elements. In this study, we identified ER and FoxA1 binding sites conserved in evolution by using Detection of LinEage-Specific Selection (DLESS) method. We also analyzed target genes close to conserved cis-regulatory elements by combining gene-expression data. A total of 7877 ER binding sites and 18,135 FoxA1 binding sites were identified in MCF-7 cells by performing an unbiased genome-wide ChIP-chip with False Discovery Rate (FDR) of 5%. Using DLESS method, we found target candidate genes closest to fully conserved cis-regulatory elements related to apoptosis according to gene ontology analysis. Furthermore, network analysis of apoptosis-related genes within 10 kb of fully conserved cis-regulatory elements was constructed using Ingenuity Pathway Analysis (IPA). Apoptosis genes in the network showed over- and under-expression in MCF-7 cell-line. And these apoptosis-related genes closest to fully conserved cis-regulatory elements in network showed strong correlation with ER in MCF-7 cells. These results elucidate that ER regulates an evolutionarily conserved apoptosis pathway. This opens up new perspectives in the apoptosis of human breast cancer from the evolution of cis-regulatory elements.

The evolution of gene regulation, the RNA universe, and the vexed questions of artefact and noise

In the ten years since the first sequencing of the human genome, much has been made of the need to look to gene regulation, and not gene number or DNA sequence, for the evolution of organismal diversity and complexity - an issue that rose to prominence, with the realization first, that the number of human genes is about the same as the number required to specify a nematode worm; and second, that the DNA of H. sapiens is roughly 96% identical to that of the chimpanzee. But the realization that the secret of evolution lies in changes in gene regulation considerably predates the revela tions of genomics. Allan Wilson and colleagues, in a paper published in 1974 [1], drew attention to the simple and striking fact that morphologically homogeneous frog species also have relatively homogeneous karyo types, whereas mammalian species, which are marked ly diverse morphologically, also show major differ ences in chromosome number and organization; changes in proteins, by contrast, are much the same for both groups. They concluded that genome organization, and by implication gene regulation, is more important for metazoan evolution than protein sequence (and cite earlier publications of EB Ford and Susumu Ohno for the same insight). The following year, Mary-Claire King and Wilson published a more detailed examination of the chromosomal distinctions between human and chimpanzee [2], arguing compellingly, without benefit of highthroughput anything, that changes in the organization of the genome, and not changes in protein-coding sequence, must account for the crucial differences between the two primates. In those pre-genomic days, the protein data were in large part immunological and electrophoretic; the analysis of genome reorganization depended on chromosome banding patterns (Giemsa banding, not FISH); and almost nothing was known of the mechanism of gene regulation in eukaryotes. The ground between then and now is covered in a recent review by Sean Carroll [3], who acknowledges Emile Zuckerkandl and Eric Davidson as early proponents of the importance of gene regulation in morphological evolution and charts the remarkable history of the development of ideas consequent on the discovery of the homeobox genes, with a strong emphasis on the evolution of cis-regulatory elements - that is to say, DNA binding sites for gene regulatory proteins - as the basis for morphological change. The argument is that DNA regulatory elements and the proteins that bind to them, often combinatorially, constitute regulatory networks that can evolve rapidly through changes to the regulatory elements, which are often modular, different modules binding different proteins characteristic of distinct differentiated states of a cell. The gene regulatory proteins can also change, of course, but are generally more highly conserved than their binding sites. Tuch et al. [4] have published a short and pellucid overview of the essential points and principles of this schema, in the context of recent evidence on how such regulatory circuits can become rewired in yeast. Beyond regulatory proteins