An in vivo system to study genetic and epigenetic regulation of DNA replication at the Igh locus

Abstract

Different DNA replication origins are utilized to replicate the Igh locus in different types of cells, suggesting they are under developmental control. To investigate what genetic and/or epigenetic factors play a key role in the selective activation of DNA replication origins in mammalian cells, we established a versatile system to alter the genetic and epigenetic status in vivo at the endogenous Igh locus in mouse ES cells by a combination of homologous recombination, Recombinase Mediated Cassette Exchange (RMCE) and specific DNA-protein interactions. This approach not only allows us to insert different DNA sequences at the same site without leaving a selective marker gene, but also provides an in vivo method to target any protein of interest to a specific site in the genome. With this system, we inserted 14 copies of Gal4 UAS site followed by an EGFP reporter gene between constant region genes Calpha and C&egr; in the Igh locus. Gal4-VP16 expression strongly activated the transcription of the EGFP reporter gene and increased histone acetylation nearby. However, no origins were activated in the Swa1 DNA segment containing the cassette. Although Gal4-VP16 can enhance the activity of the DNA replication origin in vitro or in yeast, it is not known whether it can activate the potential DNA replication origin in mammalian cells in its native chromatin context. Our study is the first to show that localized historic acetylation and transcription activation are not sufficient to create DNA replication origins at the endogenous Igh locus in the mouse ES cells. In contrast, insertion of a 10.7 kb DNA fragment, which is cloned from the replication initiation zone 3' to the Igh locus and enriched with matrix attachment region (MAR) and AT track, between Calpha and C&egr; generated novel origins. Our results suggest that large scale changes in higher order chromatin organization seem to play a more critical role than local histone modification and transcription for the activation of developmentally regulated DNA replication origins at the Igh locus.