Introduction enzymes (DUBs) 12,13. It is still unclear, however,

Introduction

The regulation of genetic information in a eukaryotic cell is crucial to proper cellular function. Methylation of DNA is one method the cell may use to regulate the expression of genetic information. One of the enzymes responsible for DNA methylation is DNA methyltransferase 1 (Dnmt1). During DNA replication Uhrf1, a protein which interacts with Dnmt1, binds to the chromatin via a hemi-methyl DNA binding site which promotes Dnmt1 binding to chromatin. Once Dnmt1 methylates the hemi-methylated DNA, Uhrf1 and Dnmt1 dissociate from the DNA. In previous studies, it was suggested that when Uhrf1 to recruited Dnmt1 the presence of ubiquitylated histones H3 was essential. It was also suggested that ubiquitylated histones H3 regulate Dnmt1 during the maintenance of DNA methylation10,11. Protein deubiquitylation is highly regulated by many deubiquitylating enzymes (DUBs) 12,13. It is still unclear, however, 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. 

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This study aims to provide the mechanism and purpose of Usp7 in relation to DNA methylation. It is possible to study the involvement of Usp7 on maintaining DNA methylation in mammals and Xenopus by inhibiting or decreasing Usp7 levels and observing the resultant effect on DNA methylation. The results of the study have suggested that H3 histone deubiquitylation by Usp7 is important to maintenance of DNA methylation14.

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

In order to determine if DUBs are involved in the rapid turnover of ubiquitylated histones H3 and DNA methylation experiments were conducted using DUB inhibitors. The inhibitor Ubiquitin vinyl sulfone (Ub-VS) was placed into a solution containing Xenopus egg extract and the maintenance of DNA methylation was observed using sperm chromatin that had 3H-S-Adenosyl-L-methionine incorporated. It was observed that the chromatin binding of Dnmt1 and Uhrf1 were suppressed and DNA methylation was inhibited. When free ubiquitin was introduced the ubiquitylation of H3 was restored, when Uhrf1 was present, however this did not restore DNA methylation.  Similar results were observed when another DUB inhibitor was used, PR-619. 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 chromatin14.  

The Dnmt1 complex stability and interaction in relation to Usp7

To determine if Usp7 is part of the Dnmt1/ Uhrf1 complex on the chromatin, and whether Usp7 contributes it’s the stability experimentations were conducted. When a mass spectrometry analysis was done on a immunopurified Dnmt1 complex, from Xenopus egg extract, it was observed that Usp7 was part of said complex. This demonstrates the binding of Usp7 in the Dnmt1 complex and suggests Usp7’s involvement in DNA methylation. In another experiment, however, it was also demonstrated that Usp7 is not necessary for Dnmt1 and Uhrf1 binding to chromatin. In the experiment a proteasomal inhibitor, MG132, was introduced to sperm chromatin and resulted in Dnmt1 and Uhrf1 binding normally to chromatin. This suggests that Usp7 does not stabilize the Dnmt1 complex by preventing proteasomal degradation.

 

The relation of Usp7 to Ubiquitylated histones H3 levels

To determine the relationship between Usp7 and ubiquitylated histones H3 several experiments were conducted. The first experiment aimed to observe how depletion of Usp7 effected the levels of ubiquitylated histones H3. This was done by placing sperm chromatin under a depletion of a mock-, USP7-, or USP7/Uhrf1- environment.  The accumulation of ubiquitylated histones H3 was confirmed using a tagged His6-ubiquitin and anti-histone H3 antibodies on an immunoblotting. The depletion of Usp7 resulted in the increased levels of ubiquitylated histones H3. When Usp7 and Uhrf1 were depleted, there was no accumulation of ubiquitylated histones H314. This result suggests a relationship between Usp7 and ubiquitylated histones H3. Another experiment was also conducted in order 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. It was the observed that xUsp7 and hUsp7 deubiquitylated ubiquitylated histones H3. 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 chromatin14.

 

 Usp7’s regulation of DNA methylation maintenance

 In order to determine if Usp7 is involved in the regulation of the maintenance of DNA Usp7 was depleted or inhibited in several experiments. In one experiment Usp7 was depleted from sperm chromatin and extracted. Then the extracts were placed in the presence of a radiolabeled S-methyl-3H-adenosyl-L-methionine and the rate of methylation was measured. The Usp7 depletion resulted in a delay in DNA methylation but not in DNA replication14.  This suggests that Usp7 is involved in DNA methylation but does not participate in DNA replication. Another experiment was done in which interphase 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 methylation14.

Discussion

This study aimed to investigate the role Usp7 played in the maintenance of DNA methylation and the nature of the relation between Usp7 and ubiquitylated histones H3. It was demonstrated that Usp7 is involved in the regulation of maintenance DNA by targeting ubiquitylated histones H3, this was dependent on the presence of Uhrf1, and interacting with the Dnmt1 complex. This is supported by several observations from experimentations. For example, when USP7 was inhibited or depleted maintenance of DNA methylation was compromised and resulted in significant accumulation of ubiquitylated histones H3. This suggested that Usp7 is involved in the turnover of ubiquitylated histones H3 in a normal cell and plays a crucial role in DNA methylation14. Consistent with this conclusion and observations made, it has been previously reported that depletion of Usp7 does reduce the efficiency of DNA methylation1,5.  

This study also provides new information on the role of Usp7 on the binding of Dnmt1 complex. This study suggested that Usp7 does not stabilize the Dnmt1 complex on chromatin by preventing proteasomal degradation as previously suggested1,2,3. This was suggested after observing Dnmt1 binding was not suppressed, but accumulated, when a Ub-VS, a DUB inhibitor, was introduced to chromatin. The suppression of Dnmt1 binding, however, appeared to be incomplete compared to the egg extraction treatment with P22077, a Usp7 inhibitor. This suggests that there is an unidentified DUB(s) sensitive to P22077 involved in the regulation of H3 histones ubiquitylation and DNA methylation. This finding opens the door to new research in the future to identify other DUBs that may be involved in DNA methylation.  

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