Vertebrate HoxB gene expression requires DNA replication

Abstract

To study the relationship between DNA replication and transcription in vivo, we investigated Hox gene activation in two vertebrate systems: the embryogenesis of Xenopus and the retinoic acid-induced differentiation of pluripotent mouse P19 cells. We show that the first cell cycles following the mid- blastula transition in Xenopus are necessary and sufficient for HoxB activation, whereas later cell cycles are necessary for the correct expression pattern. In P19 cells, HoxB expression requires proliferation, and the entire locus is activated within one cell cycle. Using synchronous cultures, we found that activation of HoxB genes is colinear within a single cell cycle, occurs during S phase and requires S phase. The HoxB locus replicates early, whereas replication is still required for maximal expression later in S phase. Thus, induction of HoxB genes occurs in a DNA replication-dependent manner and requires only one cell cycle. We propose that S-phase remodelling licenses the locus for transcriptional regulation.

Fig. 1. Inhibition of DNA replication in Xenopus embryos and Hox expression. (A) Summary of this experiment: schema showing cell-cycle stages and timing during early Xenopus development and timing of hydroxyurea and aphidicolin (HU-A) addition. MBT, mid-blastula transition; G, onset of gastrulation. (B) HU-A was added to developing Xenopus embryos before or just after the MBT at the indicated times (left) and maintained throughout development. Genomic DNA was quantitatively prepared from embryos frozen during the time course at the indicated times (top), and DNAs from 0.5 embryo equivalents were loaded on a gel and analysed by gel electrophoresis and ethidium bromide staining. NA, not applicable, since these embryos (i) at these time points are from the control group and shown in the control lanes, thus no DNA was loaded in these lanes (left) or (ii) started to disaggregate by 24 h (right). (C) Phenotype of embryos photographed at the indicated times after fertilization. (D) Total HoxB expression in replication-inhibited embryos. At the indicated times from the five different time courses, five embryos were lysed for total RNA preparation, and semiquantitative RT–PCR and Southern blotting was performed for the indicated Hox genes, the ornithine decarboxylase gene (ODC) and the genes XSox17α and DG70. Time points were (1) 5 h 30, (2) 6 h 30, (3) 7 h 30, (4) 9 h, (5) 12 h, (6) 24 h, (7) 48 h and (8) 72 h.

Fig. 2. HoxB expression in the CNS is disrupted in replication-inhibited Xenopus embryos. In situ hybridization (see Materials and methods) for Hoxb1 (left) and Hoxb9 mRNA (right) in normal (A) or replication- inhibited embryos to which hydroxyurea and aphidicolin was added at 9 h (B) or 7.5 h (C) after fertilization and maintained in the medium. Embryos were fixed at 72 h after fertilization. HoxB expression was fixed for all embryos after the same length of time of the staining reaction. The lower images are close-ups of the same embryos (left, A and C, and right B) or embryos treated in exactly the same way (left, B, and right, A and C) as those in the upper images. The arrows point to normal expression of the two Hox genes in the CNS (see text for details).

Fig. 3. Expression of all HoxB genes in P19 cells is activated during the first cell cycle after induction. (A) Schema showing the position, size, orientation and colinearity of gene transcription in the mouse HoxB locus. (B) Cell proliferation continues after retinoic acid (RA) stimulation. P19 cells were seeded at 10% confluence, incubated overnight and then either stimulated (+RA) or not (-RA) with 0.5 µM RA, and triplicate samples were trypsinized and counted. (C) HoxB activation occurs within one cell cycle. P19 cells were grown to early exponential phase, aggregated in suspension overnight, aggregates stimulated by RA, and extracts prepared during a 12 h time course for subsequent analysis by semiquantitative RT–PCR and Southern hybridization.

Fig. 4. Inhibition of DNA replication disrupts HoxB expression in P19 cells. P19 cell aggregates were treated with retinoic acid (RA) with or without aphidicolin (Aphi) and samples were lysed for semiquantitative RT–PCR at the indicated times and detected by Southern blotting using labelled probes for the relevant HoxB genes (abbreviated to b1, b2, etc.) and Cyp26 (for GAPDH, staining of the gel is shown). No expression was detected without cDNA input in the PCR (data not shown). N, non-treated P19 cell aggregates.

Fig. 5. Inhibition of DNA replication disrupts HoxB transcription in P19 cells. (A) Aphidicolin does not destabilize HoxB mRNA. P19 aggregates were stimulated by retinoic acid (RA) for 18 h and duplicate samples were incubated either with actinomycin D (ActD) alone (left) or actinomycin D and aphidicolin (+Aphi, right) for an 8 h time course. Samples were prepared for semiquantitative RT–PCR and Southern hybridization at the indicated times. (B) Experiment performed as in Figure 4, but cells were lysed for nuclear transcript purification, followed by semiquantitative RT–PCR and Southern blotting. N, non-treated P19 aggregates.

Fig. 6. Passage through DNA replication, rather than mitosis, is required for correct HoxB expression. (A) Exponentially growing (exp) P19 cell aggregates were pre-synchronized with 2 mM thymidine (+Thy) for 15 h and then released for 6 h (-Thy) before adding retinoic acid (RA) for a further 15 h, either alone or in combination with aphidicolin (Aphi), cycloheximide (CHX) or NG97. (B) Progression through the cell cycle was checked by flow cytometry. (C) Southern blots of semiquantitative RT–PCR products from the same experiment. N, non-treated aggregates; RA alone, or with aphidicolin (A), cycloheximide (C) or NG97 (97).

Fig. 7. Temporal colinearity of HoxB expression occurs within one cell cycle, and most HoxB genes are activated during S phase. Aggregates of P19 cells were synchronized by a double thymidine (Thy) block, as schematized in (A) and described in Materials and methods. Progression through the cell cycle was checked by flow cytometry. RA, retinoic acid. (B) Samples were taken at 1 h intervals for semiquantitative RT–PCR and Southern blotting.

Fig. 8. The HoxB locus replicates early in untreated P19 cells. Exponentially growing monolayer P19.6 cells were treated with a 45 min bromodeoxyuridine (BrdU) pulse and fixed and sorted by flow cytometry (A) into four S-phase compartments (1–4). (B) Replicated DNA was recovered by anti-BrdU immunoprecipitation from these four samples (1–4) and the equivalent quantity of unlabelled P19 genomic DNA (G). Semiquantitative PCR was performed using primers corresponding to the Hox genes shown on the right (b1–b9), or a region of the Hox locus at a set distance from Hoxb9 (–40 kb and –12 kb) or regions of the adenosine deaminase (ADA) and β-globin loci.

Fig. 9. S-phase transitions in dependence of HoxB expression on DNA replication. Parallel cultures of double thymidine synchronized P19 cell aggregates (syn) were released into G₂ for 6 h and retinoic acid (RA) was added, corresponding to time zero (0 h), to one population and bromodeoxyuridine (BrdU) to the other. The time course was then followed for 10 h. (A) Summary of experiment (aphi, aphidicolin). (B) Left: samples were also withdrawn and fixed for flow cytometry every 2 h to monitor cell-cycle progression (ctl, exponentially growing control cells). Right: summary of replication timing analysis for the loci shown, as determined by quantitative anti-BrdU chromatin immunoprecipitation and PCR, from parallel time course with samples every 30 min. (C) RA was added at 0 h to all samples; aphidicolin was then added to samples at half-hour intervals within the 10 h time course, and RNA was extracted from all samples at the end of the 10 h, for analysis of gene expression by semiquantitative RT–PCR and Southern blotting. N, non-RA-treated cells.

Fig. 10. Inhibition of histone deacetylation and methylation affects HoxB expression but does not mimic retinoic acid (RA) induction. (A) Southern blots of semiquantitative RT–PCR products from an experiment in which P19 aggregates were treated with RA alone, trichostatin (TSA) alone or both (R+TSA). N, non-treated cells. Time points were (1) 1 h, (2) 3 h, (3) 9 h and (4) 24 h. No products were detectable in the PCR blank (data not shown). (B) An experiment similar to that in (A) to test whether TSA could restore normal HoxB expression to P19 cells blocked by aphidicolin. Aggregates were treated with RA alone or in combination with aphidicolin (+Aph), TSA or both. Time points were (1) 2 h, (2) 8 h and (3) 24 h. N, non-treated cells.