Genetic background of phenotypic variation

A noteworthy feature of the living world is its bewildering variability. A key issue in several biological disciplines is the achievement of an understanding of the hereditary basis of this variability. Two opposing, but not necessarily irreconcilable conceptions attempt to explain the underlying mechanism. The gene function paradigm postulates that phenotypic variance is generated by the polymorphism in the coding sequences of genes. However, comparisons of a great number of homologous gene and protein sequences have revealed that they predominantly remained functionally conserved even across distantly related phylogenic taxa. Alternatively, the gene regulation paradigm assumes that differences in the cis-regulatory region of genes do account for phenotype variation within species. An extension of this latter concept is that phenotypic variability is generated by the polymorphism in the overall gene expression profiles of gene networks. In other words, the activity of a particular gene is a system property determined both by the cis-regulatory sequences of the given genes and by the other genes of a gene network, whose expressions vary among individuals, too. Novel proponents of gene function paradigm claim that functional genetic variance within the coding sequences of regulatory genes is critical for the generation of morphological polymorphism. Note, however, that these developmental genes play direct regulatory roles in the control of gene expression.     

<Emphasis Type="Italic">Cis</Emphasis>-regulatory change and expression divergence between duplicate genes formed by genome duplication of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

As an index of functional divergence, expression divergence between duplicate gene copies has been observed and correlated with protein coding sequence divergence and bias in gene functional classes. However, the changes in the cis-regulatory region of the duplicate genes which is thought to have important role in expression divergence, has not been explored on the genome-wide scale. We analyzed functional genomics data for a large number of duplicated gene pairs formed by ancient polyploidy events in Arabidopsis thaliana. The divergence in cis-regulatory regions between two copies is positively correlated with the magnitude difference of expression. Moreover, we find that highly expressed duplicate gene pairs have a more diverged cis-regulatory region than weakly expressed gene pairs. We also show that the correlation between expression functional constraint and protein functional constraint is different in old and young duplicate pairs. Our results suggest that cis-regulatory sequence divergence contributes to the expression divergence of duplicate genes formed by genome-wide du-plication. Cis-regulatory region diverges faster in highly expressed duplicate pairs. The diversify selection strengths that act on cis-regulatory region and protein coding region are negatively correlated in young duplicate pairs under expression con-straint.     

Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head

The comparison of Hox genes between vertebrates and their closest invertebrate relatives (amphioxus and ascidia) highlights two derived features of Hox genes in vertebrates: duplication of the Hox gene cluster, and an elaboration of Hox expression patterns and roles compared with non-vertebrate chordates. We have investigated how new expression domains and their associated developmental functions evolved, by testing the cis-regulatory activity of genomic DNA fragments from the cephalochordate amphioxus Hox cluster in transgenic mouse and chick embryos. Here we present evidence for the conservation of cis-regulatory mechanisms controlling gene expression in the neural tube for half a billion years of evolution, including a dependence on retinoic acid signalling. We also identify amphioxus Hox gene regulatory elements that drive spatially localized expression in vertebrate neural crest cells, in derivatives of neurogenic placodes and in branchial arches, despite the fact that cephalochordates lack both neural crest and neurogenic placodes. This implies an elaboration of cis-regulatory elements in the Hox gene cluster of vertebrate ancestors during the evolution of craniofacial patterning.     

A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.     

Polymorphism of human Ia antigens generated by reciprocal intergenic exchange between two DR beta loci

Class II molecules encoded by the human major histocompatibility complex (MHC) are involved in regulating T-cell response to antigens. The mechanisms for generating polymorphism in products of the MHC have been studied extensively for both the murine H-2 and the human HLA complex. Such studies indicate that point mutations plus selection have a major role in the generation of polymorphisms of class I and class II MHC genes. However, a non-reciprocal gene conversion mechanism has been proposed to explain several examples of clustered sequence variation in MHC genes. In all these examples, the proposed gene conversion event is unidirectional; that is, one of the two interacting genes acts as sequence donor and the other as sequence recipient. No examples of potential reciprocal genetic exchange (as occurs in the fungal system), in which the two interacting genes act as both donor and recipient of gene fragments, have been found in the MHC system or in other multigene families of higher organisms. We sequenced two different HLA-DR beta complementary DNAs from each of two different cells all expressing the same serologically defined determinant (DR2) but different T-cell-recognized (Dw) specificities (Dw12 and MN2). Sequence comparisons of these four cDNA clones (and two DR beta amino-acid sequences from the DR2-Dw2 subtype) suggest that new coding sequences for DR beta molecules in the DR2 haplotypes are potentially generated by reciprocal intergenic exchange.     

A gene regulatory network controlling the embryonic specification of endoderm

Specification of endoderm is the prerequisite for gut formation in the embryogenesis of bilaterian organisms. Modern lineage labelling studies have shown that in the sea urchin embryo model system, descendants of the veg1 and veg2 cell lineages produce the endoderm, and that the veg2 lineage also gives rise to mesodermal cell types. It is known that Wnt/β-catenin signalling is required for endoderm specification and Delta/Notch signalling is required for mesoderm specification. Some direct cis-regulatory targets of these signals have been found and various phenomenological patterns of gene expression have been observed in the pre-gastrular endomesoderm. However, no comprehensive, causal explanation of endoderm specification has been conceived for sea urchins, nor for any other deuterostome. Here we propose a model, on the basis of the underlying genomic control system, that provides such an explanation, built at several levels of biological organization. The hardwired core of the control system consists of the cis-regulatory apparatus of endodermal regulatory genes, which determine the relationship between the inputs to which these genes are exposed and their outputs. The architecture of the network circuitry controlling the dynamic process of endoderm specification then explains, at the system level, a sequence of developmental logic operations, which generate the biological process. The control system initiates non-interacting endodermal and mesodermal gene regulatory networks in veg2-derived cells and extinguishes the endodermal gene regulatory network in mesodermal precursors. It also generates a cross-regulatory network that specifies future anterior endoderm in veg2 descendants and institutes a distinct network specifying posterior endoderm in veg1-derived cells. The network model provides an explanatory framework that relates endoderm specification to the genomic regulatory code.     

Novel developmental specificity in the nervous system of transgenic animals expressing growth hormone fusion genes

The development of methods for introducing foreign genes into the germ line of mice provides an approach for studying mechanisms underlying inducible and developmental gene regulation. Transgenic animals expressing foreign genes have thus been used to test models of the role played by specific DNA sequences in determining cell-specific expression. Results from these experiments suggest that tissue-specific expression is the consequence of a cis-acting regulatory sequence. However, these results do not exclude the possibility that cell-specific expression of some genes might be 'coded' by combinations of regulatory elements. We have previously described the production of transgenic mice from eggs microinjected with metallothionein-I/growth hormone (MGH) fusion genes, and now demonstrate that the juxtaposition of sequences from two different genes can be deciphered by cells to generate novel tissue specificities. Although expression of the endogenous metallothionein and growth hormone genes has not been detected in neuronal cells, transgenic mice clearly express an MGH fusion gene in a restricted subset of neurones. These results suggest a model in which tissue-specific patterns of expression of certain genes are determined by combinations of cis-acting regulatory sequences.     

Nucleotide sequence of chicken c-myb complementary DNA and implications for myb oncogene activation

Avian myeloblastosis virus (AMV), like other acute transforming viruses, arose by recombination between its helper virus and host cellular sequences. The latter sequences, termed v-myb, are responsible for the oncogenic properties of the virus. AMV causes acute myeloblastic leukaemia in chickens and transforms a specific class of haematopoietic cells in vitro, but does not induce morphological transformation of cultured fibroblasts, suggesting that only a restricted target-cell population is responsive to its transforming gene product. The normal cellular counterpart of v-myb, c-myb, is highly conserved and is present in all vertebrate and some invertebrate species examined. DNA rearrangements and altered expression of the myb oncogene have been reported in mouse lymphoid tumours and human myeloid and colon tumours. The mechanism of activation of the cellular proto-oncogenes is thought to involve the structural alteration of the coding regions that result in either the synthesis of an altered gene product or the enhanced expression of a proto-oncogene caused by alterations in its regulatory elements. To distinguish between these two mechanisms, we have cloned and sequenced the chicken c-myb complementary DNA and compared it with that of v-myb sequences. We demonstrate that during the transduction of the cellular sequences and/or viral passage a substantial portion of the coding region of the c-myb gene has been lost from both the 5' and 3' ends, resulting in the generation of a truncated gene product that mediates the transforming function of the virus.     

Human-specific loss of regulatory DNA and the evolution of human-specific traits

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.     

Mapping and analysis of chromatin state dynamics in nine human cell types

Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.