RESUMO
In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains.
Assuntos
Replicação do DNA/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Origem de Replicação , Animais , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Camundongos , Proteínas Recombinantes de Fusão/análise , Ativação TranscricionalRESUMO
In mammalian cells, the replication of tissue-specific gene loci is believed to be under developmental control. Here, we provide direct evidence of the existence of developmentally regulated origins of replication in both cell lines and primary cells. By using single-molecule analysis of replicated DNA (SMARD), we identified various groups of coregulated origins that are activated within the Igh locus. These origin clusters can span hundreds of kilobases and are activated sequentially during B cell development, concomitantly with developmentally regulated changes in chromatin structure and transcriptional activity. Finally, we show that the changes in DNA replication initiation that take place during B cell development, within the D-J-C-3'RR region, occur on both alleles (expressed and nonexpressed).
