Identification of a functional transposon insertion in the maize domestication gene tb1

Genetic diversity created by transposable elements is an important source of functional variation upon which selection acts during evolution. Transposable elements are associated with adaptation to temperate climates in Drosophila, a SINE element is associated with the domestication of small dog breeds from the gray wolf and there is evidence that transposable elements were targets of selection during human evolution. Although the list of examples of transposable elements associated with host gene function continues to grow, proof that transposable elements are causative and not just correlated with functional variation is limited. Here we show that a transposable element (Hopscotch) inserted in a regulatory region of the maize domestication gene, teosinte branched1 (tb1), acts as an enhancer of gene expression and partially explains the increased apical dominance in maize compared to its progenitor, teosinte. Molecular dating indicates that the Hopscotch insertion predates maize domestication by at least 10,000 years, indicating that selection acted on standing variation rather than new mutation.     

A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.     

Chromatin profiling using targeted DNA adenine methyltransferase

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology. We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes. HP1 binds predominantly to pericentric genes and transposable elements. GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs. A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin. High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.     

Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids

We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse-hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The -2log10P support interval for the ceQTLs was <150 kb, containing an average of <2-3 genes. We identified 29,769 trans ceQTLs with -log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome.     

Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data

Much of a cell's activity is organized as a network of interacting modules: sets of genes coregulated to respond to different conditions. We present a probabilistic method for identifying regulatory modules from gene expression data. Our procedure identifies modules of coregulated genes, their regulators and the conditions under which regulation occurs, generating testable hypotheses in the form 'regulator X regulates module Y under conditions W'. We applied the method to a Saccharomyces cerevisiae expression data set, showing its ability to identify functionally coherent modules and their correct regulators. We present microarray experiments supporting three novel predictions, suggesting regulatory roles for previously uncharacterized proteins.     

Comparative analysis of the genome sequences of Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative beta-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica-like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.     

Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.     

Disruption of an imprinted gene cluster by a targeted chromosomal translocation in mice

Genomic imprinting is an epigenetic process in which the activity of a gene is determined by its parent of origin. Mechanisms governing genomic imprinting are just beginning to be understood. However, the tendency of imprinted genes to exist in chromosomal clusters suggests a sharing of regulatory elements. To better understand imprinted gene clustering, we disrupted a cluster of imprinted genes on mouse distal chromosome 7 using the Cre/loxP recombination system. In mice carrying a site-specific translocation separating Cdkn1c and Kcnq1, imprinting of the genes retained on chromosome 7, including Kcnq1, Kcnq1ot1, Ascl2, H19 and Igf2, is unaffected, demonstrating that these genes are not regulated by elements near or telomeric to Cdkn1c. In contrast, expression and imprinting of the translocated Cdkn1c, Slc22a1l and Tssc3 on chromosome 11 are affected, consistent with the hypothesis that elements regulating both expression and imprinting of these genes lie within or proximal to Kcnq1. These data support the proposal that chromosomal abnormalities, including translocations, within KCNQ1 that are associated with the human disease Beckwith-Wiedemann syndrome (BWS) may disrupt CDKN1C expression. These results underscore the importance of gene clustering for the proper regulation of imprinted genes.     

Reverse engineering of regulatory networks in human B cells

Cellular phenotypes are determined by the differential activity of networks linking coregulated genes. Available methods for the reverse engineering of such networks from genome-wide expression profiles have been successful only in the analysis of lower eukaryotes with simple genomes. Using a new method called ARACNe (algorithm for the reconstruction of accurate cellular networks), we report the reconstruction of regulatory networks from expression profiles of human B cells. The results are suggestive a hierarchical, scale-free network, where a few highly interconnected genes (hubs) account for most of the interactions. Validation of the network against available data led to the identification of MYC as a major hub, which controls a network comprising known target genes as well as new ones, which were biochemically validated. The newly identified MYC targets include some major hubs. This approach can be generally useful for the analysis of normal and pathologic networks in mammalian cells.     

Detection of regulatory variation in mouse genes

Functional polymorphism in genes can be classified as coding variation, altering the amino-acid sequence of the encoded protein, or regulatory variation, affecting the level or pattern of expression of the gene. Coding variation can be recognized directly from DNA sequence, and consequently its frequency and characteristics have been extensively described. By contrast, virtually nothing is known about the extent to which gene regulation varies in populations. Yet it is likely that regulatory variants are important in modulating gene function: alterations in gene regulation have been proposed to influence disease susceptibility and to have been the primary substrate for the evolution of species. Here, we report a systematic study to assess the extent of cis-acting regulatory variation in 69 genes across four inbred mouse strains. We find that at least four of these genes show allelic differences in expression level of 1.5-fold or greater, and that some of these differences are tissue specific. The results show that the impact of regulatory variants can be detected at a significant frequency in a genomic survey and suggest that such variation may have important consequences for organismal phenotype and evolution. The results indicate that larger-scale surveys in both mouse and human could identify a substantial number of genes with common regulatory variation.