The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

Abstract

The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G 1 and early S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promoter had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic spacer was markedly suppressed (but not eliminated) in early S phase. Furthermore, replication of the locus required virtually the entire S period, as opposed to the usual 3-4 h. However, restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promoterless variants revealed that initiation occurs at a low level in early S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin.

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading

“…Thus, changes in the transcription program will likely affect ORC localization and origin usage. Indeed, numerous examples exist in the literature of origin activity being modulated by transcription (Danis et al 2004;Saha et al 2004;Norio et al 2005).…”

Genome-wide depletion of replication initiation events in highly transcribed regions

“…In several biological contexts, however, transcription appears to suppress replication initiation. This occurs during differentiation of Xenopus (Hyrien et al 1995) and Sciara (Lunyak et al 2002) embryos, at the Chinese hamster Dhfr locus (Saha et al 2004), and at the murine HoxB locus (Gregoire et al 2006). Conversely, at the human IGH locus (Zhou et al 2002) replication initiates in an expanded region after the onset of transcription; and on human (Gray et al 2007) and murine (Rowntree and Lee 2006) X chromosomes, replication initiates from distinct origins regardless of transcriptional status.…”

Discrete functional elements required for initiation activity of the Chinese hamster dihydrofolate reductase origin beta at ectopic chromosomal sites

“…The observation that elements >3 kb from the ori-beta initiation site are critical for ori-beta activity is perhaps surprising given that a 1.2 kb fragment from the human LaminB2 origin is sufficient for ectopic initiation activity [21]. However, such a distal effect is not unprecedented given that deletion of the distal hamster DHFR promoter affects replication initiation in the entire downstream 55 kb intergenic region [46].…”