The long-range goal is to define the role of chromosome architecture, and in particular interaction with the nuclear matrix, in the regulation of replication origin activity in mammalian cells. The model system is the CHOC 400 cell line, in which one allele of the dihydrofolate reductase (DHFR) locus has been amplified 1,000 times. The amplicons are 240 kb in length and are arranged in tandem at three chromosomal locations. Each amplicon is characterized by two convergently transcribed genes (DHFR and 2BE2121) separated by a 55 kb spacer region. Replication can initiate anywhere within the intergenic spacer, but at least regions (termed ori-beta, ori-beta, and ori-gamma) are somewhat preferred. Ori-beta/ori-beta and ori-gamma are separated by approximately 20 kb and straddle a permanent matrix attachment region (MAR). Both direct and circumstantial evidence suggest that attachment of the intergenic region to the nuclear matrix at the intergenic MAR is required for origin function: 1) deletion of a 7 kb fragment containing the MAR converts the DHFR domain from early- to late-replicating; 2) only 10-15 percent of amplicons are actually affixed to the nuclear matrix at the intergenic MAR and only approximately 15 percent of amplicons support active initiation events in any one cell cycle; 3) ori- beta and ori-gamma (and possibly other sites in the intergenic spacer) display prominent nuclease hypersensitive sites, but only in the 15 percent of amplicons that partitions with the nuclear matrix. We propose to precisely define the MAR, to identify the proteins that are responsible for affixing the MAR to the matrix, and to analyze how attachment to the matrix affects chromatin architecture.
Specific aims are: 1) to characterize in detail the intergenic cis-element in the knock-out cell line whose loss results in loss of early origin activity in the intergenic region, using a novel homologous recombination and selection strategy; 2) to identify, isolate and characterize the trans- acting factors that interact with this critical cis-element; 3) to determine whether the DHFR origins that fire in a given cell cycle are the same ones that are attached to the nuclear matrix; 4) to determine when during the cell cycle the nuclease hypersensitive sites at ori-beta and ori-gamma appear, and whether these sites partition with the matrix because they themselves are attached or because they reside in fragments that are attached to the matrix through the intergenic MAR.
| Mesner, L D; Hamlin, J L; Dijkwel, P A (2003) The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation. Proc Natl Acad Sci U S A 100:3281-6 |
