Uncovering new functions for histone variants: a role for H2A.Z in silencing retrotransposons
Date
2016
Authors
Shen, Qian
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Abstract
Eukaryotic genomes must be, on the one hand, highly compacted by
wrapping their DNA around histones to form nucleosomes while on
the other, still remain accessible to the transcriptional
machinery in a developmental and cell-type specific manner.
Various epigenetic-based mechanisms exist that can regulate DNA
accessibility ensuring the proper regulation of gene
transcription. One epigenetic modification in particular that
impacts all aspects of genome function and organization is the
replacement of canonical histones with their variant forms. Among
the histone variants that are most extensively studied is the
essential and evolutionary conserved variant, H2A.Z. Recent
studies have revealed many layers of regulation and complex
functions of H2A.Z in modulating gene expression. Notably, H2A.Z
has the ability to both activate and repress transcription but it
remains unresolved how H2A.Z affects gene expression in these
opposing ways. At a genome-wide level, H2A.Z is found at active
transcription start sites, inactive promoters and of relevance to
this thesis, it is also found at satellite repeats including
those present at constitutive heterochromatin and the centromere.
To gain new insights into the function of H2A.Z and how it can
perform these opposing roles, this thesis has examined the
transcriptional role of H2A.Z in regulating two different types
of RNAPII transcribed elements: the retrotransposon, LINE-1 and a
coding gene important for T-cell development, Cd69.
Long Interspersed Nucleotide Element-1 (LINE-1) is one of most
impactful and still active transposable elements (TEs) that
occupy 18%-20% of mammalian genomes. Given that H2A.Z is targeted
to repetitive mouse DNA sequences found at heterochromatin and
the centromere, which is necessary for chromatin compaction, I
wondered whether this role might extend to other repetitive DNA
elements. Specifically, I explored the role of H2A.Z in LINE-1
transcription in four different cell types that were
undifferentiated (mouse trophoblast stem cells (TSCs)),
differentiated (trophoblast giant cells (TGCs) and mouse L929
cells) and committed (mouse embryonic fibroblasts (MEFs)).
The results showed significant variability in H2A.Z occupancy
relative to H2A at LINE-1s in these different cell types.
Moreover, the enrichment of H2A.Z was inversely correlated with
LINE-1 RNA expression. Specifically, relative high levels of
LINE-1 transcripts were observed in TSCs compared to MEFs and
L929 cells and this was correlated with a lower level of H2A.Z in
the promoter and coding region of the LINE-1 element.
Furthermore, when TSCs were differentiated into TGCs in vitro,
there was a dramatic reduction in LINE-1 expression and this
repression was positively correlated with a gain of H2A.Z on
LINE-1s. These observations suggested that H2A.Z was a repressor
of LINE-1 transcription.
To directly test this hypothesis, a H2A.Z knockdown system was
successfully established in both L929 and TSCs. Indeed, the loss
of H2A.Z activated LINE-1 transcription in both L929 cells and
TGCs following its differentiation from TSCs. These data shows
unequivocally that H2A.Z is a repressor of LINE-1 expression.
Unexpectedly though, the inhibition of H2A.Z expression in TSCs
decreased LINE-1 expression rather than increasing it. Further
investigations revealed that the level of acetylated H2A.Z
(acH2A.Z) at the promoter region of LINE-1s differed between TSCs
and TGCs. Significantly, there was a higher level of acH2A.Z at
LINE-1s in TSCs compared to TGCs consistent with acH2A.Z being a
mark of active transcription and the higher level of
transcription seen in TSCs. These results demonstrate for the
first time that H2A.Z is a regulator of LINE-1 transcription and
whether it activates or represses these repetitive elements is
dependent upon the abundance and the acetylation state of H2A.Z.
To further explore the role of H2A.Z in the regulation of gene
transcription, I chose to study the activation of the Cd69
promoter during T-cell development. The advantage of studying
T-cell development is that it is possible to obtain a pure
population of T-cells at different stages of development. The
Cd69 gene was of interest because it is a surface marker that
accompanies induction or repression through different stages of
thymocyte development. This enabled an investigation of when
during development H2A.Z is recruited to the Cd69 promoter, and
the relationship between this recruitment with gene activation
and the engagement of RNA polymerase II (RNAPII). Surprisingly,
the data demonstrated that H2A.Z was present on the Cd69 promoter
early in development when it is inactive and prior to the
recruitment of RNAPII. Furthermore, H2A.Z was subsequently
removed at a developmental stage prior to the binding of RNAPII
suggesting again that H2A.Z may be a repressor of gene
transcription during development. In conclusion, this thesis has
revealed new unexpected functions for H2A.Z in the regulation of
LINE-1 and Cd69 transcription.
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Keywords
LINE-1s, H2A.Z, RNAPII, Trophoblast stem cells, CD69
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