Identification of Differentially Methylated Regions (DMRs) between Embryonic Stem Cells (ESCs) and Embryonic Germ Cells (ESCs) by DNA methylation studies
Epigenetic is the study of heritable changes that are not caused by the changes in the DNA sequence. It involves post–translational modifications of histones and cytosine modifications, resulting in long–term alterations in the transcriptional potential of a cell that are not necessarily heritabl...
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| Format: | Thesis |
| Language: | English |
| Published: |
2018
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| Subjects: | |
| Online Access: | http://eprints.usm.my/56859/1/Khoo%20Jo%20Lynn-24%20pages.pdf |
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| Summary: | Epigenetic is the study of heritable changes that are not caused by the
changes in the DNA sequence. It involves post–translational modifications of
histones and cytosine modifications, resulting in long–term alterations in the
transcriptional potential of a cell that are not necessarily heritable. One of the major
epigenetic events, known as genomic imprinting, is a phenomenon in which some
genes are expressed in a parent–of–origin–specific manner. To date, over 100 genes
are known to be regulated by genomic imprints, many of which have significant roles
for embryogenesis, placental formation and brain development. In this study,
embryonic stem cells (ESCs) and embryonic germ cells (EGCs) were used. ESCs are
pluripotent stem cells derived from inner cell mass (ICM) of pre–implantation
embryos called blastocyst, while EGCs are pluripotent stem cells originated from
primordial germ cells (PGCs). Although genomes of ICM cells are globally
hypomethylated, primary DNA methylation imprints are retained in ICM and its
derivatives, i.e. ESCs. Therefore, monoallelic imprinted gene expression will be
established in ESCs upon differentiation. In contrast, PGCs colonised in the gonads
have undergone global DNA demethylation, by which DNA methylation imprints are
supposed to be erased. Thus, ESCs and EGCs are highly similar cells except for one
aspect, that is, the imprinting status. Comparison of ESCs with EGCs may uncover epigenetic modifications required for the establishment of imprinted gene expression.
Toward this end, ESCs and EGCs were differentiated by embryoid body formation
and harvested every other day, being verified by qPCR and allele specific expression.
Methylation of imprints were confirmed to be retained in ESCs but not EGCs at both
undifferentiated and differentiated state by Reduced Representative Bisulfite
Sequencing (RRBS) and SureSelectXT Methyl–Seq Combined with Post–Bisulfite
Adaptor Tagging (SureSelect–PBAT) methods. A number of differentially methylated
regions (DMRs) other than known imprinted regions were identified for the first time
between ESCs and EGCs. Interestingly, DMRs that are hypermethylated in EGCs
while being hypomethylated in ESCs were also able to be identified. Although some
of these DMRs were technically validated, methylation levels showed were different
from that of RRBS and SureSelect–PBAT method. Nonetheless, these DMRs still
exhibited the same hypermethylated/hypomethylated state in ESCs/EGCs. Gene
expression studies by RNA–seq demonstrated that only a small proportion of genes
with 2–or more–fold difference in expression between ESCs and EGCs were able to
be correlated with their methylation levels, as most of the DMRs were identified at
intron and intergenic regions, not promoter regions. |
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