Biomedical subjects

Chii Mei Lin

Publications and source records attributed to Chii Mei Lin.

4 recordsLinked to original sources

PubMed2006

Cooperative sequence modules determine replication initiation sites at the human beta-globin locus.

The human beta globin locus contains two adjacent replicators, each capable of initiating DNA replication when transferred from its native locus to ectopic sites. Here, we report a detailed analysis of the sequence requirements for replication initiation from these replicators. In both replicators, initiation required a combination of an asymmetric purine:pyrimidine sequence and several AT-rich stretches. Modules from the two replicators could combine to initiate replication. AT-rich sequences were essential for replicator activity: a low frequency of initiation was observed in DNA fragments that included a short stretch of AT-rich sequences, whereas inclusion of additional AT-rich stretches increased initiation efficiency. By contrast, replication initiated at a low level without the asymmetric purine:pyrimidine modules but they were required in synergy to achieve efficient initiation. These data support a combinatorial model for replicator activity and suggest that the initiation of DNA replication requires interaction between at least two distinct sequence modules.

Base Sequence↗

PubMed2006

DNA methylation supports intrinsic epigenetic memory in mammalian cells.

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

Animals↗

PubMed2005

The human beta-globin locus control region can silence as well as activate gene expression.

Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.

Animals↗

PubMed2003

Dynamic alterations of replication timing in mammalian cells.

BACKGROUND: The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated. RESULTS: We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human beta-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the beta-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene. CONCLUSIONS: Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.

Animals↗