DNA methylation is an epigenetic modification that plays an important role in development
and differentiation. The patterns of DNA methylation are largely established in early
embryogenesis and maintained during development. Abnormal DNA methylation patterns have
been associated with many human diseases, including cancer. Despite its importance, little is
currently known about the mechanisms that determine DNA methylation patterns throughout the
genome. To shed light on the molecular mechanisms that regulate DNA methylation, this study
investigates whether DNA methylation patterns are established and maintained normally when
human DNA is placed into a heterologous murine environment as opposed to its natural,
endogenous chromosomal environment.
Here, a previously generated transgenic mouse model, containing 117 kb of human DNA
bearing the human α-globin cluster and all of its known regulatory elements, was analysed. The
pattern of DNA methylation of the endogenous human α-globin cluster was compared with that of
the transgenic cluster in the background of mouse embryonic stem cells (ESCs) and tissues. It
was found that, although the normal human DNA methylation pattern was largely established and
maintained in a mouse background, the region immediately around the human α-globin genes
themselves is generally less methylated in mouse compared to human ESCs. It was found that
regions adjacent and up to 2kb from the CpG islands (CGIs), so-called CGI shores, were
unusually hypomethylated: this seems to be the result of an extension of CGIs in humanised
mouse (hm) ESCs compared to human (h) ESCs. Furthermore, this hypomethylation appeared to
increase during development in both erythroid and non-erythoid cells.
To identify any cis-regulatory sequences responsible for the hypomethylated state of
human CGI shores in the mouse, 2-4 kb human test sequences containing the CGI associated
with the human α-globin 2 (α2) gene and its adjacent hypomethylated shore were re-integrated
into the mouse α-globin locus via recombination-mediated cassette exchange (RMCE). Human
CGI shores became hypomethylated in the context of the re-integrated test sequences, indicating
that the appearance of hypomethylation is determined by the underlying human DNA sequence in
the test fragments.
In summary, the data presented here reveal that human CGIs become extended when
placed in a mouse background leading to hypomethylation of human CGI shores in the mouse
compared to the pattern of methylation at the normal endogenous human locus. These findings
suggest that species-specific factors determine DNA methylation near CGIs. The transgenic
mouse model provides an excellent system to dissect out species-specific regulation of CGI shore
methylation. Furthermore, this study lays the foundation for future experiments addressing the role of DNA methylation in regulating human gene expression in the murine context, and examining
the validity of transgenic mouse models for the study of human gene regulation.This thesis is currently not available on ORA