Pavla Navratilova

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881–885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770–775; Frayling TM, et al. (2007) Science 316:889–894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing β-cells and glucagon-producing α-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

Systematic human/zebrafish comparative identification of cis-regulatory activity around vertebrate developmental transcription factor genes☆

Pan-vertebrate developmental cis-regulatory elements are discernible as highly conserved noncoding elements (HCNEs) and are often dispersed over large areas around the pleiotropic genes whose expression they control. On the loci of two developmental transcription factor genes, SOX3 and PAX6, we demonstrate that HCNEs conserved between human and zebrafish can be systematically and reliably tested for their regulatory function in multiple stable transgenes in zebrafish, and their genomic reach estimated with confidence using synteny conservation and HCNE density along these loci. HCNEs of both human and zebrafish function as specific developmental enhancers in zebrafish. We show that human HCNEs result in expression patterns in zebrafish equivalent to those in mouse, establishing zebrafish as a suitable model for large-scale testing of human developmental enhancers. Orthologous human and zebrafish enhancers underwent functional evolution within their sequence and often directed related but non-identical expression patterns. Despite an evolutionary distance of 450 million years, one pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting that multiple regulatory inputs are required to achieve robust and precise complex expression patterns exhibited by developmental genes.

Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons

Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity.

Morphogenesis underlying the development of the everted teleost telencephalon

BackgroundAlthough the mechanisms underlying brain patterning and regionalization are very much conserved, the morphology of different brain regions is extraordinarily variable across vertebrate phylogeny. This is especially manifest in the telencephalon, where the most dramatic variation is seen between ray-finned fish, which have an everted telencephalon, and all other vertebrates, which have an evaginated telencephalon. The mechanisms that generate these distinct morphologies are not well understood.ResultsHere we study the morphogenesis of the zebrafish telencephalon from 12 hours post fertilization (hpf) to 5 days post fertilization (dpf) by analyzing forebrain ventricle formation, evolving patterns of gene and transgene expression, neuronal organization, and fate mapping. Our results highlight two key events in telencephalon morphogenesis. First, the formation of a deep ventricular recess between telencephalon and diencephalon, the anterior intraencephalic sulcus (AIS), effectively creates a posterior ventricular wall to the telencephalic lobes. This process displaces the most posterior neuroepithelial territory of the telencephalon laterally. Second, as telencephalic growth and neurogenesis proceed between days 2 and 5 of development, the pallial region of the posterior ventricular wall of the telencephalon bulges into the dorsal aspect of the AIS. This brings the ventricular zone (VZ) into close apposition with the roof of the AIS to generate a narrow ventricular space and the thin tela choroidea (tc). As the pallial VZ expands, the tc also expands over the upper surface of the telencephalon. During this period, the major axis of growth and extension of the pallial VZ is along the anteroposterior axis. This second step effectively generates an everted telencephalon by 5 dpf.ConclusionOur description of telencephalic morphogenesis challenges the conventional model that eversion is simply due to a laterally directed outfolding of the telencephalic neuroepithelium. This may have significant bearing on understanding the eventual organization of the adult fish telencephalon.

Genomic regulatory blocks in vertebrates and implications in human disease

Despite a recent explosion in the production of vertebrate genome sequence data and large-scale efforts to completely annotate the human genome, we still have scant knowledge of the principles that built vertebrate genomes in evolution, and of genome architecture and its functional significance. We review approaches using bioinformatics, zebrafish transgenesis, and recent findings in the molecular basis of gene regulation and tie these in with mechanisms for the maintenance of long-range conserved synteny across all vertebrate genomes. Specifically, we discuss the recently discovered genomic regulatory blocks which we argue are principal units of vertebrate genome evolution and serve as the foundations onto which evolutionary innovations are built through sequence evolution and insertion of new cis-regulatory elements. We subsequently discuss how these arrangements relate to common human heritable diseases and their significance in disease causality.

Regulatory divergence of the duplicated chromosomal loci sox11a/b by subpartitioning and sequence evolution of enhancers in zebrafish

We used the classic example of the duplicated zebrafish sox11a and -b loci to test the duplication, degeneration, complementation (DDC) model of genome evolution through whole genome duplication. While recent reports have demonstrated sub-partitioning of regulatory sequences in duplicated regions, a comparison of the regulatory capabilities of extant regulatory sequences derived from ancient ancestral elements has been scarce. Consistent with the DDC model, we find that ancestral regulatory elements distributed over several hundred kb were lost in either one or the other zebrafish duplicate, leading to subpartitioning. However, regulatory sequences kept as duplicates near both sox11 co-orthologs diverged in sequence from each other and from human elements and in the regulatory patterns they drive in transgenic zebrafish. Evolutionary analysis of the loci suggested that both zebrafish protein coding sox11 orthologs have been maintained by purifying selection, and have evolved at comparable rates, indicative of non-diverged protein functions. The duplicated regulatory elements, conversely, evolved with different divergence rates and degrees of subfunctionalization. These data show that regulatory evolution of gene expression patterns occurred both through differential loss as well as through regulatory sequence evolution in zebrafish versus human genomes.

Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses

Greying with age in horses is an autosomal dominant trait, characterized by hair greying, high incidence of melanoma and vitiligo‐like depigmentation. Previous studies have revealed that the causative mutation for this phenotype is a 4.6‐kb intronic duplication in STX17 (Syntaxin 17). By using reporter constructs in transgenic zebrafish, we show that a construct containing two copies of the duplicated sequence acts as a strong enhancer in neural crest cells and has subsequent melanophore‐specific activity during zebrafish embryonic development whereas a single copy of the duplicated sequence acts as a weak enhancer, consistent with the phenotypic manifestation of the mutation in horses. We further used luciferase assays to investigate regulatory regions in the duplication, to reveal tissue‐specific activities of these elements. One region upregulated the reporter gene expression in a melanocyte‐specific manner and contained two microphthalmia‐associated transcription factor (MITF) binding sites, essential for the activity. Microphthalmia‐associated transcription factor regulates melanocyte development, and these binding sites are outstanding candidates for mediating the melanocyte‐specific activity of the element. These results provide strong support for the causative nature of the duplication and constitute an explanation for the melanocyte‐specific effects of the Grey allele.

Distinct core promoter codes drive transcription initiation at key developmental transitions in a marine chordate

BackgroundDevelopment is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates.ResultsOur TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box.ConclusionsOur results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa.

Trans-splicing of mRNAs links gene transcription to translational control regulated by mTOR

In phylogenetically diverse organisms, the 5’ ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. Here, we quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, showing that the SL sequence permits nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continues to filter-feed, but arrests growth until favorable conditions return. Upon release from such conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. Our results demonstrate a role for trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under limiting conditions and suggest an innovative strategy for rapid evolution of mTOR targets in genomes of metazoans whose reproduction is tightly linked to nutritional cues.

Erratum to: Morphogenesis underlying the development of the everted teleost telencephalon.

It has been brought to the Publisher’s attention that Additional file 2 (Figure S1) was uploaded incorrectly during publication of the original version of this article [1]. The corrected figure can be found below.