Francisco Antequera

Structure, function and evolution of CpG island promoters

Mammalian promoters belong to two different categories in terms of base composition and DNA methylation. In humans and mice, approximately 60% of all promoters colocalize with CpG islands, which are regions devoid of methylation that have a higher G+C content than the genome average, while the rest have a methylation pattern and base composition indistinguishable from bulk DNA. Recent comparative studies between both organisms have refined our understanding of how CpG island promoters are organized in terms of protein-DNA interactions and patterns of expression. In addition, the finding that DNA replication initiates at CpG islands in vivo suggests that their distinctive properties could be a consequence of such activity and opens the possibility of a coordinated regulation of transcription and replication. These new data shed light on the origin and evolution of the CpG islands and should contribute to improving methods for promoter prediction in the human and mouse genomes.

Initiation of DNA replication at CpG islands in mammalian chromosomes

CpG islands are G+C‐rich regions ∼1 kb long that are free of methylation and contain the promoters of many mammalian genes. Analysis of in vivo replication intermediates at three hamster genes and one human gene showed that the CpG island regions, but not their flanks, were present in very short nascent strands, suggesting that they are replication origins (ORIs). CpG island‐like fragments were enriched in a population of short nascent strands from human erythroleukaemic cells, suggesting that islands constitute a significant fraction of endogenous ORIs. Correspondingly, bulk CpG islands were found to replicate coordinately early in S phase. Our results imply that CpG islands are initiation sites for both transcription and DNA replication, and may represent genomic footprints of replication initiation.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus

The INK4/ARF locus encodes three tumour suppressors (p15INK4b, ARF and p16INK4a) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RDINK4/ARF). Targeted and localized RNA-interference-induced heterochromatinization of RDINK4/ARF results in transcriptional repression of the locus, revealing that RDINK4/ARF is a relevant transcriptional regulatory element. Cdc6 is overexpressed in human cancers, where it might have roles in addition to DNA replication. We have found that high levels of Cdc6 result in RDINK4/ARF-dependent transcriptional repression, recruitment of histone deacetylases and heterochromatinization of the INK4/ARF locus, and a concomitant decrease in the expression of the three tumour suppressors encoded by this locus. This mechanism is reminiscent of the silencing of the mating-type HM loci in yeast by replication factors. Consistent with its ability to repress the INK4/ARF locus, Cdc6 has cellular immortalization activity and neoplastic transformation capacity in cooperation with oncogenic Ras. Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16INK4a. We conclude that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RDINK4/ARF element.

Genome-wide distribution of DNA replication origins at A+T-rich islands in Schizosaccharomyces pombe

Genome‐wide analysis of replication dynamics requires the previous identification of DNA replication origins (ORIs). However, variability among the ORIs makes it difficult to predict their distribution across the genome on the basis of their sequence. We report here that ORIs in Schizosaccharomyces pombe coincide with discrete chromosomal A+T‐rich islands of up to 1 kb long that are characterized by a distinctive A+T content that clearly differentiates them from the rest of the genome. Genome‐wide analysis has enabled us to identify 384 of these regions, which predicts the position of most ORIs in the genome, as shown by functional replication analyses. A+T‐rich islands occur at the mating locus, centromeres and subtelomeric regions at a density that is approximately fourfold higher than elsewhere in the genome, which suggests a link between the origin recognition complex (ORC) and transcriptional silencing in these regions. The absence of consensus elements in A+T‐rich islands implies that different sequences can target the ORC to different ORIs.

Organization of DNA replication origins in the fission yeast genome

Eukaryotic DNA replication initiates at multiple points along the chromosomes known as replication origins (ORIs). We have developed a strategy to identify ORIs directly from replication intermediates in the fission yeast Schizosaccharomyces pombe. Mapping of a selection of the novel ORIs onto the genome reveals their preferential localization at intergenic regions upstream from genes. These results are supported by the observation that a large proportion of regions overlapping gene promoters contain active ORIs. Mapping of the genomic ars1 replication origin at nucleotide resolution shows that replication initiates at a defined position immediately upstream from the hus5+ promoter. Deletion analysis indicates that the regulatory elements required to initiate transcription and replication lie in close proximity, suggesting a possible relationship between both processes in vivo.

Increased Recombination Intermediates and Homologous Integration Hot Spots at DNA Replication Origins

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.

Species-specific organization of CpG island promoters at mammalian homologous genes

An essential issue derived from the sequencing of the human and other genomes is the identification of gene regulatory elements. Using in vivo footprinting and expression analysis, here we show that mouse and human CpG island promoters at homologous genes have a completely different organization in terms of size and binding of transcription factors. Despite these species‐specific differences, a unifying picture emerges from the precise confinement of protein–DNA interactions between the 5′ boundary of the CpG islands and the transcription initiation site. This finding allows direct localization of promoters on genomic sequences and reveals a very high rate of variation and evolutionary divergence of mammalian regulatory regions. Our results also show that CpG island promoters associated with tissue‐specific genes, such as the human α‐globin, are bound by ubiquitous factors that allow a constitutive low level of expression in many cell types.

Genomic specification and epigenetic regulation of eukaryotic DNA replication origins

Identification of DNA replication origins (ORIs) at a genome‐wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.

Overreplication of short DNA regions during S phase in human cells

DNA replication origins (ORI) are regulatory regions from which the genome is replicated once every cell cycle. A widely used method for their identification in mammalian chromosomes relies on quantitative PCR of DNA nascent strands across candidate regions. We developed a new high-resolution PCR strategy to localize ORIs directly on total unfractionated human DNA. The increase in sensitivity provided by this approach has revealed that a short region of approximately 200-base-pair overlapping well-characterized replication origins undergoes several rounds of replication, coinciding with their specific time of activation during S phase. This process generates a population of discrete dsDNA fragments detectable as free molecules in preparations of total DNA in normally proliferating cells. Overreplicated regions have precise boundaries at the edge of the nucleosome-free gap that encompasses the transcription initiation sites of CpG island promoters. By itself, active transcription does not induce overreplication but does stimulate it at ORIs associated with promoters. The coincidence in time and space between the overproduction of short DNA fragments and ORI activity predicts the precise localization of thousands of ORIs in the human genome and uncovers a previously unnoticed step in the initiation of DNA replication.

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe.

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)‐rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T‐rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low‐efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals.