The role of transcription in the activation of a Drosophila amplification origin

Abstract

The mechanisms that underlie metazoan DNA replication initiation, especially the connection between transcription and replication origin activation, are not well understood. To probe the role of transcription in origin activation, we exploited a specific replication origin in Drosophila melanogaster follicle cells, ori62, which coincides with the yellow-g2 transcription unit and exhibits transcription-dependent origin firing. Within a 10-kb genomic fragment that contains ori62 and is sufficient for amplification, RNA-sequencing analysis revealed that all detected RNAs mapped solely to the yellow-g2 gene. To determine whether transcription is required in cis for ori62 firing, we generated a set of tagged yellow-g2 transgenes in which we could prevent local transcription across ori62 by deletions in the yellow-g2 promoter. Surprisingly, inhibition of yellow-g2 transcription by promoter deletions did not affect ori62 firing. Our results reveal that transcription in cis is not required for ori62 firing, raising the possibility that a trans-acting factor is required specifically for the activation of ori62. This finding illustrates that a diversity of mechanisms can be used in the regulation of metazoan DNA replication initiation.

Keywords: DNA replication; gene amplification; promoter deletion; replication initiation; yellow-g2.

Figure 1

Left, Model of transcription-dependent origin activation of DAFC-62D. DAFC-62D exhibits two rounds of origin firing, the first at stage 10B and the second at stage 13 of egg chamber development, which are interspersed by transcription of the yellow-g2 gene. In the presence of the RNA polymerase II inhibitor α-amanitin, stage 13 origin firing is specifically blocked (Xie and Orr-Weaver 2008). Right, Transcription-dependent origin firing is unique to DAFC-62D, as exemplified by normal origin firing of the comparable DAFC-34B in the presence of α-amanitin (Kim and Orr-Weaver 2011).

Figure 2

Analysis of transcripts within the 10-kb amplification-sufficient fragment of DAFC-62D (black bar) from two biological replicates. Total, non-poly(A)-selected RNA was isolated from 16C follicle cell nuclei, which are enriched for amplification stages. RNAs were sequenced and mapped to the Drosophila dm3 genome. The chr3L:2,270,000-2,288,000 region is shown, and the 5′ ends of each gene are depicted.

Figure 3

Characterization of the full-length tagged yellow-g2 transgene. (A) Structure of the 16.2-kb [DAFC-62D-10kb] P-element transposon construct. (B) Diagram of the full-length, 1.5-kb yellow-g2 transgene. A 21-bp tag was inserted in the 3′ end of the yellow-g2 coding sequence. Extent of the full yellow-g2 promoter is unknown. (C) Levels of transgenic yellow-g2 transcripts relative to total yellow-g2 transcripts isolated from stage 12 egg chambers in either wild-type or su(Hw) mutant backgrounds. Fold amplification of the endogenous 62D locus (62D Endo) and the [DAFC-62D-10kb] transposon was measured in the wild-type (D) and the su(Hw) mutant (E) backgrounds. Error bars represent standard deviation of the mean of the biological triplicates.

Figure 4

Characterization of the yellow-g2 promoter deletion transgenes. (A) Diagrams of the full-length, 214-bp promoter deletion, and 1226-bp promoter deletion yellow-g2 transgenes. The region deleted is shown in white. The 1226-bp deletion is not to scale. The extent of the full yellow-g2 promoter is not known. Levels of transgenic yellow-g2 transcripts relative to total yellow-g2 transcripts isolated from stage 12 egg chambers were determined for the [DAFC-62D-Δ214] (B) and [DAFC-62D-Δ1226] (C) transgenic lines in either wild-type or su(Hw) mutant backgrounds. (D) Fold amplification of the endogenous 62D locus (62D Endo) and the [DAFC-62D-Δ214] transposon in the wild-type and the su(Hw) mutant backgrounds. (E) Fold amplification of the endogenous 62D locus (62D Endo) and the [DAFC-62D-Δ1226] transposon in the wild-type and the su(Hw) mutant backgrounds. Error bars represent standard deviation of the mean of the biological triplicates.