Asynchronous Replication, Mono-Allelic Expression, and Long Range Cis-Effects of ASAR6

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.
Author Summary Mammalian chromosomes are duplicated every cell cycle during a precise temporal DNA replication program. Thus, every chromosome contains regions that are replicated early and other regions that are replicated late during each S phase. Most of the genes, present in two copies on homologous chromosomes, replicate synchronously during each S phase. Exceptions to this rule are genes located on X chromosomes, genetically imprinted genes, and genes subject to allelic exclusion. Thus, all mono-allelically expressed genes are subject to asynchronous replication, where one allele replicates before the other. Perhaps the best-studied example of asynchronous replication in mammals occurs during X inactivation in female cells. A large non-coding RNA gene called XIST, located within the X inactivation center, controls the transcriptional silencing and late replication of the inactive X chromosome. We have identified a locus on human chromosome 6 that shares many characteristics with XIST. This chromosome 6 locus encodes a large intergenic non-coding RNA gene, ASAR6, which displays random mono-allelic expression, asynchronous replication, and controls the mono-allelic expression of other genes on chromosome 6. Our work supports a model in which all mammalian chromosomes contain similar cis-acting loci that function to ensure proper chromosome replication, mitotic condensation, mono-allelic expression, and stability of individual chromosomes.