Determinants of nucleosome organization in primary human cells

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.     

Topological domains in mammalian genomes identified by analysis of chromatin interactions

The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.     

Mutation rates differ among regions of the mammalian genome

In the traditional view of molecular evolution, the rate of point mutation is uniform over the genome of an organism and variation in the rate of nucleotide substitution among DNA regions reflects differential selective constraints. Here we provide evidence for significant variation in mutation rate among regions in the mammalian genome. We show first that substitutions at silent (degenerate) sites in protein-coding genes in mammals seem to be effectively neutral (or nearly so) as they do not occur significantly less frequently than substitutions in pseudogenes. We then show that the rate of silent substitution varies among genes and is correlated with the base composition of genes and their flanking DNA. This implies that the variation in both silent substitution rate and base composition can be attributed to systematic differences in the rate and pattern of mutation over regions of the genome. We propose that the differences arise because mutation patterns vary with the timing of replication of different chromosomal regions in the germline. This hypothesis can account for both the origin of isochores in mammalian genomes and the observation that silent nucleotide substitutions in different mammalian genes do not have the same molecular clock.     

Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA

Tandem-repetitive minisatellite regions in vertebrate DNA frequently show substantial allelic variation in the number of repeat units. This variation is thought to arise through processes such as unequal crossover or replication slippage. We show here that the spontaneous mutation rate to new length alleles at extremely variable human minisatellites is sufficiently high to be directly measurable in human pedigrees. The mutation rate at different loci increases with variability in accord with the neutral mutation/random drift hypothesis, and rises to 5% per gamete for the most unstable human minisatellite isolated. Mutations are sporadic, occur with similar frequencies in sperm and oocytes, and can involve the gain or loss of substantial numbers of repeat units, consistent with length changes arising primarily by unequal exchange at meiosis. Germline instability must therefore be taken into account when using hypervariable loci as genetic markers, particularly in pedigree analysis and parenthood testing.     

Frequent mtDNA mutations and its role in gastric carcinogenesis

In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occurred in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 coding genes and D-loop control region, which was 36.7%, 26.7% and 30% respectively. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ. Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complexⅠ, especially ND3, ND4 and ND5 genes, might contribute to human gastric carcinogenesis.     

Patterns of somatic mutation in human cancer genomes

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.     

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.     

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.     

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.