<p>Mammalian development is
orchestrated by global transcriptional changes, which drive cellular
differentiation, giving rise to diverse cell types. The mechanisms that mediate
the temporal control of early differentiation can be studied using embryonic
stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In
these stem cells, differentiation signals induce transcriptional repression of
genes that maintain pluripotency (PpG) and activation of genes required for
lineage specification. Expression of PpGs is controlled by these genes’
proximal and distal regulatory elements, promoters and enhancers, respectively.
Previously published work from our laboratory
showed that during
differentiation of ESCs, the repression of PpGs is accompanied by enhancer
silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this
complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation
followed by a gain of DNA methylation mediated by the DNA methyltransferase,
Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC
differentiation leads to their incomplete repression. In cancer, abnormal
expression of PpG is commonly observed. Our studies show that in
differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain
substantial expression concomitant with an absence of Lsd1-mediated H3K4me1
demethylation at their respective enhancers. The continued presence of H3K4me1
blocks the downstream activity of Dnmt3a, leading to the absence of DNA
methylation at these sites. The absence of Lsd1 activity at PpG enhancers
establishes a “primed” chromatin state distinguished by the absence of DNA
methylation and the presence of H3K4me1. We further established that the
activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially
repressed post-differentiation. Our data reveal that sustained expression of
the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing
mechanism. This generates an aberrant “primed” enhancer state, which is susceptible
to activation and supports tumorigenicity. </p>
<p>As differentiation proceeds and
multiple layers of cells are produced in the early embryo, the inner cells are
depleted of O<sub>2</sub>, which triggers endothelial cell differentiation. These
cells form vascular structures that allow transport of O<sub>2</sub> and nutrients to cells. Using
ESC differentiation to endothelial cells as a model system, studies covered in
this thesis work elucidated a mechanism by which the transcription factor
Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial
differentiation and formation of vascular structures. Our data show that
Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes
in response to endothelial differentiation induction. This defect was shown to
be the result of the elevated expression of the stemness factor Cbp/p300-interacting
transactivator 2 (Cited2)
at the onset of differentiation. The improper expression of Cited2 sequesters
histone acetyltransferase p300 from depositing active histone modifications at
the regulatory elements of angiogenesis-specific genes that, in turn, impedes
their activation. </p>
<p>Besides the discovery of
epigenetic mechanisms that regulate gene expression during differentiation, our
studies also include development of a sensitive method to identify activities
of a specific DNA methyltransferase at genomic regions. In mammals, DNA
methylation occurs at the C5 position of cytosine bases. The addition of this
chemical modification is catalyzed by a family of enzymes called DNA methyltransferases
(Dnmts). Current methodologies, which determine the distribution of Dnmts or
DNA methylation levels in genomes, show the combined activity of multiple Dnmts
at their target sites. To determine the activity of a particular Dnmt in
response to an external stimulus, we developed a method, Transition State
Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps
catalytically active Dnmts at their transition state with the DNA substrate.
Our goal is to produce a strategy that would enable the determination of the
direct genomic targets of specific Dnmts, creating a valuable tool for studying
the dynamic changes in DNA methylation in any biological process.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/9033839 |
| Date | 02 August 2019 |
| Creators | Lama Abdullah Alabdi (7036082) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/MOLECULAR_MECHANISMS_THAT_GOVERN_STEM_CELL_DIFFERENTIATION_AND_THEIR_IMPLICATIONS_IN_CANCER/9033839 |
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