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of DNA is one method the cell may use to regulate the expression of genetic
information which is crucial for cellular function. An enzyme involved in DNA
methylation is DNA methyltransferase 1 (Dnmt1) and Uhrf1, a protein which
interacts with Dnmt1, promotes Dnmt1’s binding to chromatin promotes by hemi-methylation
of DNA.  Once Dnmt1 methylates the
hemi-methylated DNA, Uhrf1 and Dnmt1 dissociate from the DNA. In previous
studies, it was suggested that ubiquitylated histones H3 regulate Dnmt1 during
the maintenance of DNA methylation by aiding Uhrf1 recruitment of Dnmt1 to
chromatin7,8. Protein deubiquitylation is highly regulated by many
deubiquitylating enzymes (DUBs)4,10 but it is still unclear if any DUBs
target ubiquitylated H3 histones. It is possible that Ubiquitin-specific
protease 7 (Usp7) targets H3 histones. In mammalian cells Usp7 has been
suggested to create a stable complex with Uhrf1 and Dnmt1 during DNA
methylation by preventing proteasomal degradation of the complex1,2.  This study aims to provide the mechanism and
purpose of Usp7 in relation to DNA methylation and ubiquitylated histones H310.

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The effect of DUBs on
ubiquitylated histones H3 and DNA methylation maintenance

determine if DUBs are involved in the turnover of ubiquitylated histones H3 and
in DNA methylation DUB inhibitors were used. The inhibitor, Ubiquitin vinyl sulfone (Ub-VS) was placed in solution with Xenopus egg
extract and the maintenance of DNA methylation was observed using 3H-S-Adenosyl-L-methionine
in sperm chromatin. It was observed that the chromatin binding of Dnmt1 and
Uhrf1 were suppressed and ubiquitylated histones H3 levels were reduced.  In order to confirm results another DUB inhibitor
was used, PR-619, with similar results observed. These results suggested that a
DUB plays a crucial role in the maintenance of DNA methylation and interacts with
ubiquitylated histones H3 to bind Dnmt1 and Uhrf1 to the chromatin10.


The Dnmt1
complex stability and interaction in relation to Usp7

To confirm Usp7 is part of the Dnmt1 complex and determine if Usp7
contributes the stability of the complex experiments were conducted. When a
mass spectrometry analysis was done on a immunopurified Dnmt1 complex, from Xenopus
egg extract, it was observed that Usp7 was present. In another experiment
using a proteasomal inhibitor, MG132, it was also demonstrated that Usp7 is not
necessary for Dnmt1 complex binding to chromatin. This demonstrates that Usp7
does bind to the Dnmt1 complex and thus is involved in DNA methylation, however
it does not stabilize the Dnmt1 complex by preventing proteasomal degradation10.


Interaction of Usp7 with ubiquitylated
histones H3

determine if Usp7 targets ubiquitylated histones H3 several experiments were conducted.

The first experiment demonstrated how depletion of Usp7 effected the levels of ubiquitylated
histones H3 by placing sperm chromatin under a depletion of a Usp7.  The depletion of Usp7 resulted in the
increased levels of ubiquitylated histones H3 which was confirmed using a
tagged His6-ubiquitin and anti-histone
H3 antibodies on an immunoblotting. This
result suggests a interaction between Usp7 and ubiquitylated histones H3. To
determine whether Usp7 functioned as a DUB for ubiquitylated histone H3. This
was done by placing immunopurified xUsp7 (from the egg extract) and hUsp7
(expressed in insects) with histone H3 in vitro and the deubiquitylated ubiquitylated
histones H3 was observed. The results suggest that Usp7 does function as a DUB
for ubiquitylated histone H3, which was consistent with the results from the
Usp7 depletion in sperm chromatin10.

regulation of DNA methylation maintenance

 In order to determine if Usp7 is involved in
the regulation of the maintenance of DNA Usp7 was depleted in several
experiments and the rate of DNA methylation was measured using radiolabeled S-methyl-3H-adenosyl-L-methionine
in sperm chromatin. The Usp7 depletion resulted in a delay in DNA methylation
but not in DNA replication.  This
suggests that Usp7 is involved in DNA methylation but does not participate in
DNA replication. To confirm this egg extracts were
treated with P22077, a Usp7 inhibitor. The P22077 treatment noticeably suppressed
the rate of DNA methylation.  These
results support the notion that Usp7 plays an important role in DNA methylation10. 

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