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.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.