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. 2004 Dec 21;101(51):17771-6.
doi: 10.1073/pnas.0408170101. Epub 2004 Dec 10.

DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states

Eric J White  1 , Olof Emanuelsson, David Scalzo, Thomas Royce, Steven Kosak, Edward J Oakeley, Sherman Weissman, Mark Gerstein, Mark Groudine, Michael Snyder, Dirk Schübeler
Affiliations

DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states

Eric J White et al. Proc Natl Acad Sci U S A. .

Abstract

Duplication of the genome during the S phase of the cell cycle does not occur simultaneously; rather, different sequences are replicated at different times. The replication timing of specific sequences can change during development; however, the determinants of this dynamic process are poorly understood. To gain insights into the contribution of developmental state, genomic sequence, and transcriptional activity to replication timing, we investigated the timing of DNA replication at high resolution along an entire human chromosome (chromosome 22) in two different cell types. The pattern of replication timing was correlated with respect to annotated genes, gene expression, novel transcribed regions of unknown function, sequence composition, and cytological features. We observed that chromosome 22 contains regions of early- and late-replicating domains of 100 kb to 2 Mb, many (but not all) of which are associated with previously described chromosomal bands. In both cell types, expressed sequences are replicated earlier than nontranscribed regions. However, several highly transcribed regions replicate late. Overall, the DNA replication-timing profiles of the two different cell types are remarkably similar, with only nine regions of difference observed. In one case, this difference reflects the differential expression of an annotated gene that resides in this region. Novel transcribed regions with low coding potential exhibit a strong propensity for early DNA replication. Although the cellular function of such transcripts is poorly understood, our results suggest that their activity is linked to the replication-timing program.

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Figures

Fig. 1.
Fig. 1.
Karyotypic analysis of primary fibroblast cells (HFL-1) and the lymphoblastoid NC-NC cell line. Spectral karyotyping was used to identify chromosome number and integrity. No abnormality was detected on the autosomes, whereas a subfraction of the female NC-NC cells lacks the inactive X chromosome as described in ref. .
Fig. 2.
Fig. 2.
Experimental strategy. Shown are histogram plots of DNA content for each cell type. Asynchronously replicating cells were labeled with BrdUrd for 1 h and sorted by FACS for DNA content. DNA from cells in the first and last thirds of S phase was extracted, and newly replicated DNA was immunoprecipitated with α-BrdUrd antibody. Immunoprecipitated DNA was labeled with Cy3 or Cy5 and hybridized to the chromosome 22 DNA microarray.
Fig. 3.
Fig. 3.
Control experiments. (a) Autocorrelation analysis to measure the chromosomal extent of similar replication timing. The autocorrelation function is calculated for an increasing number of neighboring sequences. Positive autocorrelation exists if neighboring spots tend to be alike, which reflects replication at a similar time in S phase. The autocorrelation plot of replication-timing ratios before smoothing (see Fig. 4) is shown to identify the lengths of significantly similar (>0.05) DNA replication timing. (b) DNA replication-timing profile of HFL-1 compared with control hybridization. The DNA replication-timing profile of HFL-1 (green) is plotted with the mixed early and late S-phase DNA control (gray). Shown are the first 20 Mb of chromosome 22 and the chromosomal positions of amplicons used in single-gene controls. (c) Single-gene control PCR of early- and late-replicating regions for the HFL-1 cell type. Amplicon names refer to microarray features from which ≈400-bp fragments were amplified. In each case, the control reaction confirms early- or late-replication timing as revealed by the microarray analysis.
Fig. 4.
Fig. 4.
DNA replication profiles for HFL-1 and NC-NC. DNA replication-timing ratios (log2-transformed) were plotted according to chromosomal position. Shown is the nonrepetitive part of chromosome 22 extending from the centromere (Upper Left) to the telomere of the q arm (Lower Right). Data were smoothed by loess smoothing, and the best-fit plots are shown for HFL-1 (green line) and NC-NC (red line). The black mark along the baseline indicates regions of DNA replication timing that were present on the microarray and compared between both cell types. Associated orange hash marks indicate regions of significant difference (P < 0.05) in DNA replication timing. The percentage of chromosomal GC content is plotted as an orange line near the top of the plot as a sliding 100-kb window. The percentage of transcribed sequences for both cell types is plotted just below GC content as a sliding 100-kb window along the chromosome for HFL-1 (cyan line) and NC-NC (black line). Annotated genes that are expressed in HFL-1 and NC-NC are indicated as blue squares above (HFL-1) and below (NC-NC) the baseline. Locations of cytological G bands are indicated by gray boxes along the chromosome-position axis (22). (A larger version of this figure is available at http://array.mbb.yale.edu/chr22.)
Fig. 5.
Fig. 5.
Relation of replication timing to transcription. mRNA expression and novel TARs correlate with early DNA replication timing. Microarray features were binned in groups of 50 features with similar replication-timing ratios. The percentage of expressed features in each bin was calculated and plotted according to DNA replication-timing ratio. A logistic regression curve was fitted to the data points. Red data points represent those fragments overlapping with annotated genes and blue data points represent those fragments that do not overlap with annotated genes
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References

    1. Huberman, J. A. & Riggs, A. D. (1968) J. Mol. Biol. 32, 327-341. - PubMed
    1. Hand, R. (1978) Cell 15, 317-325. - PubMed
    1. Dutrillaux, B., Couturier, J., Richer, C. L. & Viegas-Pequinot, E. (1976) Chromosoma 58, 51-61. - PubMed
    1. Cimbora, D. M. & Groudine, M. (2001) Cell 104, 643-646. - PubMed
    1. Drouin, R., Holmquist, G. P. & Richer, C. L. (1994) Adv. Hum. Genet. 22, 47-115. - PubMed

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