Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes)

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.     

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13^GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13^lacZneo embryos. Similarly, Hoxa1^GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1^GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.     

Adenovirus-mediated regulable target gene expression in vivo

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor–ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-κB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.     

Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3′ to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.     

Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A–E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.     

Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae)

Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.