Adam Woolfe

Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development

In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.

Dynamics of Histone H3 Deposition In Vivo Reveal a Nucleosome Gap-Filling Mechanism for H3.3 to Maintain Chromatin Integrity

Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.

Mislocalization of the Centromeric Histone Variant CenH3/CENP-A in Human Cells Depends on the Chaperone DAXX

Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.

Genomic features defining exonic variants that modulate splicing

BackgroundSingle point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative.ResultsWe assessed the features of single-nucleotide genomic variants verified to cause exon skipping and compared them to a large set of coding SNPs common in the human population, which are likely to have no effect on splicing. Our findings implicate a number of features important for their ability to discriminate splice-affecting variants, including the naturally occurring density of exonic splicing enhancers and exonic splicing silencers of the exon and intronic environment, extensive changes in the number of predicted exonic splicing enhancers and exonic splicing silencers, proximity to the splice junctions and evolutionary constraint of the region surrounding the variant. By extending this approach to additional datasets, we also identified relevant features of variants that cause increased exon inclusion and ectopic splice site activation.ConclusionsWe identified a number of features that have statistically significant representation among exonic variants that modulate splicing. These analyses highlight putative mechanisms responsible for splicing outcome and emphasize the role of features important for exon definition. We developed a web-tool, Skippy, to score coding variants for these relevant splice-modulating features.

Comparative genomics using Fugu reveals insights into regulatory subfunctionalization

BackgroundA major mechanism for the preservation of gene duplicates in the genome is thought to be mediated via loss or modification of cis-regulatory subfunctions between paralogs following duplication (a process known as regulatory subfunctionalization). Despite a number of gene expression studies that support this mechanism, no comprehensive analysis of regulatory subfunctionalization has been undertaken at the level of the distal cis-regulatory modules involved. We have exploited fish-mammal genomic alignments to identify and compare more than 800 conserved non-coding elements (CNEs) that associate with genes that have undergone fish-specific duplication and retention.ResultsUsing the abundance of duplicated genes within the Fugu genome, we selected seven pairs of teleost-specific paralogs involved in early vertebrate development, each containing clusters of CNEs in their vicinity. CNEs present around each Fugu duplicated gene were identified using multiple alignments of orthologous regions between single-copy mammalian orthologs (representing the ancestral locus) and each fish duplicated region in turn. Comparative analysis reveals a pattern of element retention and loss between paralogs indicative of subfunctionalization, the extent of which differs between duplicate pairs. In addition to complete loss of specific regulatory elements, a number of CNEs have been retained in both regions but may be responsible for more subtle levels of subfunctionalization through sequence divergence.ConclusionComparative analysis of conserved elements between duplicated genes provides a powerful approach for studying regulatory subfunctionalization at the level of the regulatory elements involved.

Early evolution of conserved regulatory sequences associated with development in vertebrates.

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.

CONDOR: a database resource of developmentally associated conserved non-coding elements

BackgroundComparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal cis-regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.DescriptionHere we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: http://condor.fugu.biology.qmul.ac.uk. In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing in vivo zebrafish enhancer assays of the CNEs carried out in-house, which currently number ~100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental cis-regulation.ConclusionBy providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development.

Characterisation of conserved non-coding sequences in vertebrate genomes using bioinformatics, statistics and functional studies

We recently identified approximately 1400 conserved non-coding elements (CNEs) shared by the genomes of fugu (Takifugu rubripes) and human that appear to be associated with developmental regulation in vertebrates [Woolfe, A., Goodson, M., Goode, D.K., Snell, P., McEwen, G.K., Vavouri, T., Smith, S.F., North, P., Callaway, H., Kelly, K., Walter, K., Abnizova, I., Gilks, W., Edwards, Y.J.K., Cooke, J.E., Elgar, G., 2005. Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol. 3 (1), e7]. This study encompassed a multi-disciplinary approach using bioinformatics, statistical methods and functional assays to identify and characterise the CNEs. Using an in vivo enhancer assay, over 90% of tested CNEs up-regulate tissue-specific GFP expression. Here we review our groups research in the field of characterising non-coding sequences conserved in vertebrates. We take this opportunity to discuss our research in progress and present some results of new and additional analyses. These include a phylogenomics analysis of CNEs, sequence conservation patterns in vertebrate CNEs and the distribution of human SNPs in the CNEs. We highlight the usefulness of the CNE dataset to help correlate genetic variation in health and disease. We also discuss the functional analysis using the enhancer assay and the enrichment of predicted transcription factor binding sites for two CNEs. Public access to the CNEs plus annotation is now possible and is described. The content of this review was presented by Dr. Y.J.K. Edwards at the TODAI International Symposium on Functional Genomics of the Pufferfish, Tokyo, Japan, 3-6 November 2004.