HoxB domain induction silences DNA replication origins in the locus and specifies a single origin at its boundary

Abstract

In multicellular organisms, changes in the DNA replication programme could act to integrate differentiation with cell division in various developmental and transcriptional contexts. Here, we have addressed the use of DNA replication origins during differentiation in the HoxB domain-a cluster of nine genes developmentally regulated in a collinear manner. In undifferentiated mouse P19 cells, we detected several DNA replication origins in the 100 kb HoxB locus, indicating a relaxed origin use when the locus is transcriptionally silent. By contrast, in retinoic-acid-induced differentiated cells, when HoxB transcription is activated, a general silencing of DNA replication origins occurs in the locus except one located downstream of Hoxb1, at the 3' boundary of the HoxB domain. Silencing of the replication origins is associated with histone hyperacetylation, whereas the active Hoxb1 origin persists as a hypoacetylated island. These findings provide direct evidence for the differentiated use of origins in HoxB genes, and we suggest that this regulation might contribute to the regulated expression of HoxB genes during development.

Figure 1

DNA replication initiates at several sites in the HoxB domain in undifferentiated P19 cells. (A) Real-time quantitative PCR was performed on P19 nascent strands, using primer pairs (filled squares) located along the c-myc locus. Enrichment was calculated as fold enrichment over the background level (grey box; see the supplementary information online). The results shown are the average of three independent nascent strand preparations that were quantified twice. Error bars indicate the standard error of the mean. Genes are indicated by grey boxes. (B) Same as (A), but for the HoxB locus. kb, kilobase; NS, nascent strands.

Figure 2

Retinoic-acid-triggered differentiation silences replication origins in the HoxB locus. (A) Total RNA was collected from retinoic-acid-treated P19 cells on the indicated days. Complementary DNA was generated by reverse transcription and quantified by real-time PCR to analyse transcription of Hoxb1, Hoxb2, Hoxb4, Hoxb7, Hoxb9 and Oct4, a marker of undifferentiated cells. For each gene, quantities were calculated by setting the value obtained for the higher point to 100%. Averages and standard errors from two independent experiments are shown. (B) Untreated P19 cells (P19) and P19 cells treated with retinoic acid for 1 day (P19+RA D1) were hybridized with an antisense Hoxb1 probe and coloured by Hoechst. No signal was detected with a sense Hoxb1 probe. (C) Real-time quantitative PCR was performed on nascent strands purified from P19 cells treated with retinoic acid for 3 days, using primer pairs (filled squares) located along the c-myc locus. Enrichment was calculated as fold enrichment over the background level (grey box; see the supplementary information online). The results are the average of three independent nascent strand preparations that were quantified twice. Error bars indicate the standard error of the mean. Genes are indicated by grey boxes. (D) Same as (C), but for the HoxB domain. a.u., arbitrary units; kb, kilobase; NS: nascent strands; RA, retinoic acid.

Figure 3

Hoxb1 origin of replication is unaffected by transcription of the gene. Nascent strands (NS) were isolated from P19 cells (P19) and P19 cells treated with retinoic acid (RA) for 1 (RA D1) or 3 (RA D3) days. Real-time quantitative PCR was performed using the indicated primer pairs (filled squares), and the level of enrichment was calculated as fold enrichment over the background level (grey box; see the supplementary information online). Results are the average of three independent NS preparations that were quantified twice. Error bars indicate the standard error of the mean. Filled diamond indicates the position of the RA response element. kb, kilobase.

Figure 4

Retinoic acid treatment induces H3 and H4 acetylation at the HoxB locus. Levels of (A) H3, (B) acetylated H3 (acH3) and (C) acetylated H4 (acH4) were analysed by chromatin immunoprecipitation and real-time PCR quantification using the indicated primer pairs (filled squares) before (P19) and after (P19+RA) 3 days of RA-induced differentiation. As a reference, the acetylation level from the c-myc promoter is shown in the left subpanel. The results are the average of two independent immunoprecipitation preparations that were quantified twice. Error bars indicate the standard error of the mean. Genes are indicated by grey boxes. kb, kilobase; RA, retinoic acid.

Figure 5

Silencing of DNA replication origins in the HoxB domain during transcriptional induction. DNA replication origins can be present at several positions along the cluster of the nine HoxB genes at a stage when the domain is not transcribed. Induction of differentiation by retinoic acid leads to the progressive transcription of the nine genes, in a collinear manner. Transcriptional activation correlates with a global silencing of DNA replication origins inside the cluster. An important DNA replication origin remains at the 3′ border of Hoxb1, the first gene transcriptionally activated in the locus, suggesting that the HoxB cluster forms a single co-regulated chromatin unit.