Research Interests

Evaluation of epigenetic inheritance levels through development of a sister chromatin immunoprecipitation (sisChIP) assay

In the nucleus, although there are two to four alleles per gene (depending on the cell cycle stage), all of the molecular methods currently available for analysing chromatin structure are not designed for distinguishing between each of the four alleles. Thus, only the mean values obtained from multiple alleles are evaluated by existing methods. However, accumulating data suggest that sister alleles are not always epigenetically identical. Although we and others have established methods to examine homologous allele-specific epigenetic states using RFLPs, it is currently impossible to distinguish between identical sister chromatin strands at a molecular level.

In this project, we aim to establish a novel molecular method to evaluate the fidelity of epigenetic inheritance, by analysing histone modification of each sister allele. This system will allow us test a likely scenario that may generate asymmetric epigenetic states between post-replication sister allele. We will also examine if Dnmt1 null mutations and hypomorphic mutations in PCNA alleles perturb the inheritance of histone modifications. These experiments will clarify the link between epigenetic inheritance and DNA replication.

Evaluation of the link between error-prone epigenetic inheritance and the phenotype of daughter cells through single-cell transcript analyses 

Cell division is a process by which the parent cell gives rise to two daughter cells in every cell cycle. The primary consequence of cell division is to deliver parental genomes, which are replicated during S phase, to each daughter cell. Despite the high fidelity of the DNA replication process which assures that daughter cells carry identical genetic information, on many occasions, daughter cells exhibit distinct phenotypes. One example is the clonal heterogeneity of normal cells. For example, researchers have suggested that hematopoietic multipotent stem progenitor cells show heterogeneous and promiscuous gene expression profiles. Similar to somatic stem cells, various genes are heterogeneously expressed in ES cell cultures and in blastocysts.

The biological significance of clonal heterogeneity in normal cell populations is still unclear, but it has been assumed that it may be important for cell populations to respond to various differentiation signals while retaining their self-renewal potential. While the cell-to-cell variation may simply be “noise” caused by the stochastic nature of gene expression or a subtle difference of environment around each cell (e.g. position effect), evidence suggests that heterogeneity may be relevant to the differentiation program, and is caused by intrinsic cues.

Our working hypothesis is that clonal heterogeneity and asymmetric division, both of which are observed in various lineages, are in part driven by error-prone epigenetic inheritance during DNA replication. To address this, we will evaluate the similarity of daughter cells through single-cell RNA analyses in wild-type and mutant ES cells that lack functional DNA replication-related epigenetic modifiers.