Pan-S replication patterns and chromosomal domains defined by genome-tiling arrays of ENCODE genomic areas - PubMed (original) (raw)

Pan-S replication patterns and chromosomal domains defined by genome-tiling arrays of ENCODE genomic areas

Neerja Karnani et al. Genome Res. 2007 Jun.

Abstract

In eukaryotes, accurate control of replication time is required for the efficient completion of S phase and maintenance of genome stability. We present a high-resolution genome-tiling array-based profile of replication timing for approximately 1% of the human genome studied by The ENCODE Project Consortium. Twenty percent of the investigated segments replicate asynchronously (pan-S). These areas are rich in genes and CpG islands, features they share with early-replicating loci. Interphase FISH showed that pan-S replication is a consequence of interallelic variation in replication time and is not an artifact derived from a specific cell cycle synchronization method or from aneuploidy. The interallelic variation in replication time is likely due to interallelic variation in chromatin environment, because while the early- or late-replicating areas were exclusively enriched in activating or repressing histone modifications, respectively, the pan-S areas had both types of histone modification. The replication profile of the chromosomes identified contiguous chromosomal segments of hundreds of kilobases separated by smaller segments where the replication time underwent an acute transition. Close examination of one such segment demonstrated that the delay of replication time was accompanied by a decrease in level of gene expression and appearance of repressive chromatin marks, suggesting that the transition segments are boundary elements separating chromosomal domains with different chromatin environments.

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Figures

Figure 1.

Figure 1.

Temporal profile of replication of chromosomal segments. (A) Temporally specific versus pan-S distribution of replication for 1% of the human genome investigated in this study. (B) Raw TR50 data with a smoothed TR50 curve overlaid from the 1.9-Mb region on Chromosome 7. According to the ENCODE Consortium nomenclature, this chromosomal segment is referred to as ENm001 (

http://hgwdev.cse.ucsc.edu/ENCODE/encode.hg17.html

). (C) Smoothed TR50 data from the 1-Mb beta-globin locus (ENm009) on Chromosome 11. The lowest point in each valley indicates a site that is replicated before its adjoining segments and thus is likely to contain origins of replication. The gaps in the TR50 plots indicate the presence of repeats. In order to minimize cross-hybridization of oligonucleotides, repeat regions of the genome are not spotted on the tiling arrays. The triangle on the _X_-axis indicates the position of the known beta-globin origin.

Figure 2.

Figure 2.

Segregation of chromosomal segments with temporally specific and temporally nonspecific pattern of replication. (A) On the basis of TR50, temporally specific regions are further segregated and displayed as three tracks (UCSC Genome Browser), early-, mid-, or late-replicating, while chromosomal regions undergoing temporally nonspecific replication are highlighted under the pan-S track. The three panels in this figure show segregation of replication timing for three chromosomal segments. (Top panel) The 1.9-Mb region (ENm001) of Chromosome 7; (middle and bottom panels) examples of two 500-kb chromosomal segments from Chromosomes 16 (ENr313) and 13 (ENr132) that underwent late and pan-S replication, respectively. The FISH track in all the three panels refers to the chromosomal positions of BAC clones selected for the interphase FISH experiment shown in Figures 3 and 4. (B) Percent of temporally specific chromosomal segments replicating in early-, middle-, or late-S phase.

Figure 3.

Figure 3.

Interphase FISH for validating replication timing in HeLa. (A–C) Synchronously progressing HeLa cells were hybridized to fluorescence-labeled probes of BAC clone DNA RP11-51M22, RP11-3I14 (for early- and late-replicating areas, respectively) and RP11-88E10 (for pan-S pattern of replication). The chromosomal locations of these BACs are highlighted in Figure 2A. The percent replication at each interval of S phase is plotted against time in S phase. (D) The interallelic variation in replication for FISH data observed for each of the BAC clones mentioned above was determined by calculating the Mid-Score (detailed in Results and Supplemental Material).

Figure 4.

Figure 4.

Pan-S replication pattern is independent of cell synchronization method and aneuploidy. (A) HeLa cells blocked (by nocodazole) and released from mitosis followed by FACS for DNA content. (B,C) Interphase FISH was performed with HeLa cells synchronized with nocodazole and released. The _X_-axis represents time in S phase such that 0 h = 12 h post-release from the nocodazole block. The rest is as in Figure 3. (D) MCF10A cells released from a G1/S block with thymidine/aphidicolin followed by FACS for DNA content. (E,F) Interphase FISH with MCF10A cells synchronously progressing through S phase to determine the replication profile and Mid-Score with the chromosomal segments mentioned in Figure 3.

Figure 5.

Figure 5.

Correlation between replication time and genomic features. (A) Plot of smoothed TR50 against AT content in a 10-kb sliding window. Lowess smoothed curve done at f = 0.3 (fraction of the data included in the running local fit) is overlaid in black to show the general trend. (B–D) Histograms showing distribution of (B) CpG islands, (C) gene density, and (D) transcripts (HeLa cells) against temporal segregation of replication.

Figure 6.

Figure 6.

Replication profile demarcates chromosomal domains. (A) UCSC Genome Browser display of a 1.7-Mb region from Chromosome 21 (ENm005). This Browser picture highlights four tracks (I–IV): (I) FISH: chromosomal location of BAC clones (RP11-54F16 and RP11-79D9, from left to right) selected for the interphase FISH experiment shown in B; (II) Primers: chromosomal locations of the primers (005HM1–9, left to right) selected for ChIP assay to ascertain the histone modifications and HP1α-binding sites shown in D_–_F; (III) RefSeq: positions of all the genes in this chromosomal segment; and (IV) the contiguous TR50 profile. (B) Dual color FISH was performed with HeLa cells synchronously progressing through S phase. RP11-54F16 (from early-replicating area on left) was labeled with spectrum red dUTP, while RP11-79D9 (from late-replicating area) was labeled with spectrum green dUTP. Dual color FISH with these two BAC clones ascertained the replication time of the two regions of Chromosome 21 set 355 kb apart. (C) Plot of smoothed TR50 (_Y_-axis on left, gray) against level of transcription of genes (_Y_-axis on right, black). The two asterisk marks represent transcripts whose transcription levels exceeded the _Y_-axis limit (i.e., 2346 and 10,010 for left and right asterisks, respectively). (D–F) ChIP-PCR assay across ENm005 region (see Supplemental Table 3 for primers). PCR was performed on DNA chromatin immunoprecipitated with antibodies against methylated histones (H3 Lys4 and H3 Lys9) and HP1α. (Input) DNA control before immunoprecipitation; (IgG) ChIP with rabbit IgG was negative control for nonspecific precipitation. Forty cycles of PCR were performed for H3 Lys4 and HP1α and 30 cycles for H3 K9 di-Me. The asterisks refer to primer pairs that gave positive ChIP signal for the indicated antibodies relative to the IgG negative control. 005HM4 and 005HM5 were from the late-replicating island in ENm005.

Figure 7.

Figure 7.

Both active and repressive chromatin marks are present in a pan-S segment. (A) UCSC Genome Browser display of a 500-kb region from Chromosome 13 (ENr132). This Browser picture highlights three tracks: (I) Primers: ChIP-PCR primers (132HM1–3, left to right) to study histone modifications and HP1α-binding sites; (II) RefSeq: positions of all the genes in this chromosomal segment; and (III) the temporal segregation of replication data. (B,C) ChIP-PCR assay across ENr132 region (see Supplemental Table 3 for primers) against methylated histones (H3 Lys4 and H3 Lys9) and HP1α (as indicated).

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