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

Abstract

The human beta-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.

Figures

Figure 1.

Histone acetylation map of the human β-globin replication origin. Chromatin from human K562, 293, and proerythroblast (ProE) cells was subjected to ChIP analysis with antibodies against 2meH3K4, H4Ac, and H3Ac, and analyzed for relative enrichment by real-time PCR at 15 different sites along the β-globin domain on chromosome 11 (see map). The globin origin region, composed of two replicator subunits, RepI (I) and RepP (P) (Wang et al. 2004), is marked, as are the ε(HBE), γ(HBG1, HBG2), β-pseudo (HBBP1), δ(HBD), and β(HBB) gene sequences and the upstream LCR. Transcription is from right to left. Origin-specific histone acetylation and H3K4 methylation are characterized by a peak (highlighted by a white stripe). High-resolution ChIP-on-chip analysis for histone H3Ac in K562 cells over the origin region and the γ-globin genes can be seen in Supplemental Figure 1.

Figure 2.

Histone acetylation causes a shift to early replication. (A) FISH analysis (percentage of single dots) of BrdU-positive nuclei from transiently cultured, BrdU-labeled spleen lymphocytes (SL) or MEFs using the human globin BAC (H) and a mouse globin construct (M) as probes. Results from HAT-tethered mice are marked in red. (B) Bar graph showing the normalized values (Supplemental Material) for replication time as a percentage of S phase as measured in spleen lymphocytes (B14-L and B54-L) or MEFs (B14-M). Standard replication time markers included mouse globin (mHbb), as well as both the early (S1) and late (S2) asynchronous Snrpn alleles. The ranges of wild-type (WT) (late) and HAT (early) replication times are indicated by dotted lines. (Inset) Transgenic mice carrying a randomly integrated nonorigin fragment (NOC2) were crossed with animals containing the Gal4-CBP transgene, and spleen nuclei then subjected to FISH analysis with the pLJ168 probes.

Figure 3.

(A) Spleen lymphocytes from B14 wild-type (WT) and B14 VP16 (VP16) transgenic mice were transiently cultured and incubated with BrdU for 1 h and cell-cycle-sorted by FACS into G1, S1, S2, S3, S4, and G2 fractions. BrdU DNA was then purified from each sample and analyzed by real-time PCR using six different primer sets distributed over the human globin BAC. The results from both experiments were individually averaged over all probes (local abundance) and normalized to the peaks of standard early (Cd19 and Actb) and late (Hbb and Amy) replicating mouse genes and then mathematically adjusted to take into consideration variations between the separate FACS analyses (Supplemental Material). (B) Replicating fork directions were determined by isolating emetine-treated, BrdU-labeled cells and carrying out single-strand real-time PCR analysis on purified BrdU DNA at two loci (28K and 74K) positioned on opposite sides of the human β-globin origin (Supplemental Material). The ratio of normalized backward to forward synthesis is shown. Leading strand synthesis in the rightward direction shows a high ratio of backward/forward PCR synthesis, while left-hand leading strand synthesis gives an opposite result.

Figure 4.

Histone deacetylation causes a shift to late replication. (A) FISH analysis (percentage of single dots) of BrdU-positive nuclei from transiently cultured, BrdU-labeled erythroid cells using the human globin BAC (H) and a mouse globin construct (M) as probes. Results from HDAC2 mice are marked in red. (B) Bar graph showing the normalized values (Supplemental Material) for replication time as a percentage of S phase in erythroblasts (B14-E and B54-E). Standard replication time markers include mouse globin (mHbb), as well as both the early (S1) and late (S2) asynchronous Snrpn alleles. The ranges of wild-type (WT) (early) and HDAC2 (late) replication times are indicated by dotted lines.