An Origin of DNA Replication in the Promoter Region of the Human Fragile X Mental Retardation (FMR1) Gene

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 ∼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.[Keywords: Replication origins; CpG islands; promoters; transcription; cell cycle; chromatin structure] Supplemental material is available at http://www.genesdev.org.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Overreplication of short DNA regions during S phase in human cells

Gómez¹,

Antequera²

2008

“…This change appears to occur even though ␤-globin transcription remains relatively high in these cells (see Materials and Methods). Forced histone deacetylation (Coffee et al 1999) may also be responsible for the dramatic shift to late replication seen at the FMR1 locus in Fragile X syndrome as a result of de novo DNA methylation on origin sequences (Gray et al 2007) located in the gene promoter.…”

Section: Histone Modification Controls Replication Timing Genes and Devmentioning

confidence: 99%

DNA replication timing of the human β-globin domain is controlled by histone modification at the origin

Goren¹,

Tabib²,

Hecht³

et al. 2008

130

113

The human ␤-globin genes constitute a large chromosomal domain that is developmentally regulated. In nonerythroid cells, these genes replicate late in S phase, while in erythroid cells, replication is early. The replication origin is packaged with acetylated histones in erythroid cells, yet is associated with deacetylated histones in nonerythroid cells. Recruitment of histone acetylases to this origin brings about a transcription-independent shift to early replication in lymphocytes. In contrast, tethering of a histone deacetylase in erythroblasts causes a shift to late replication. These results suggest that histone modification at the origin serves as a binary switch for controlling replication timing.Supplemental material is available at http://www.genesdev.org.Received December 20, 2007; revised version accepted March 17, 2008. Studies in both yeast and animal cells demonstrate that the time at which replication origins fire during S phase is established by a preset marking system that operates in early G1 at about the same point that nuclear structure is restored following cell division (Raghuraman et al. 1997;Dimitrova and Gilbert 1999). This epigenetic signal may involve histone modification, as suggested by genetic manipulation experiments in yeast showing that normally early replicating origins can actually be made to fire aberrantly in late S phase simply by causing forced local histone deacetylation (Vogelauer et al. 2002). Whereas in yeast cells each origin replicates at an invariable fixed time within S phase, replication timing in higher eukaryotes is often subject to developmental regulation, and this is accomplished by altering the firing schedule of fixed origins. The human ␤-globin domain represents a good example of this phenomenon. Genetic and biochemical studies have demonstrated that this entire ∼100-kb region is copied by employing a single bidirectional origin that fires early during S phase in erythroid cells, but is converted to a late replication mode in all nonerythroid cells (Kitsberg et al. 1993). Nothing, however, is known about how this timing profile is set up at the molecular level and, in particular, whether this is dependent on local transcription (Gilbert 2002). Results and DiscussionIn order to assess whether this epigenetic switch may be mediated by changes in histone modification, we used chromatin immunoprecipitation (ChIP) analysis to map the mean histone acetylation pattern over the entire ␤-globin domain in different cell types (Fig. 1). In proerythroblast cells, we observed a large enrichment of histone H3 and H4 acetylation over the ␤-globin promoter and coding sequence in correlation with the transcription profile of this gene in these erythroid cells. Mapping studies indicate that the replication origin in this region is actually composed of two separate functional units (Wang et al. 2004), and it appears from our data that these sequences (see map) are also prepackaged with a combination of hyperacetylated histones and me-H3(K4) in the proerythroblast cells (F...

“…To validate this approach, we determined if peaks are enriched in small nascent strands that can only be detected proximal to active origins (Aladjem et al 1998). We isolated nascent DNA fragments from logarithmically growing cells by alkaline gel electrophoresis (Gray et al 2007). Within this fraction, we enrich for a control sequence of a previously described origin (Supplemental Fig.…”

Section: Structure and Distribution Of Replication Domainsmentioning

confidence: 99%

“…Nascent strand DNA in a size range of 1000 to 2000 bp was isolated from logarithmically growing Kc cells by alkaline gel electrophoresis (Gray et al 2007). Genomic DNA from Kc cells in G 2 phase (isolated by FACS) was used as a control.…”

Section: Nascent Strand Analysismentioning

confidence: 99%

Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome

Schwaiger¹,

Stadler²,

Bell³

et al. 2009

146

265

Duplication of eukaryotic genomes during S phase is coordinated in space and time. In order to identify zones of initiation and cell-type-as well as gender-specific plasticity of DNA replication, we profiled replication timing, histone acetylation, and transcription throughout the Drosophila genome. We observed two waves of replication initiation with many distinct zones firing in early-S phase and multiple, less defined peaks at the end of S phase, suggesting that initiation becomes more promiscuous in late-S phase. A comparison of different cell types revealed widespread plasticity of replication timing on autosomes. Most occur in large regions, but only half coincide with local differences in transcription. In contrast to confined autosomal differences, a global shift in replication timing occurs throughout the single male X chromosome. Unlike in females, the dosage-compensated X chromosome replicates almost exclusively early. This difference occurs at sites that are not transcriptionally hyperactivated, but show increased acetylation of Lys 16 of histone H4 (H4K16ac). This suggests a transcription-independent, yet chromosome-wide process related to chromatin. Importantly, H4K16ac is also enriched at initiation zones as well as early replicating regions on autosomes during S phase. Together, our study reveals novel organizational principles of DNA replication of the Drosophila genome and suggests that H4K16ac is more closely correlated with replication timing than is transcription.[Keywords: Chromatin; Drosophila; microarray; replication] Supplemental material is available at http://www.genesdev.org.