450+ experts on 30 subjects ready to help you just now
Starting from 3 hours delivery
Remember! This is just a sample.
You can get your custom paper by one of our expert writers.Get custom essay
121 writers online
In eukaryotes DNA methylation plays an important role in the transcriptional regulation of genes, silencing of transposable elements and X-chromosome inactivation to name a few. DNA is methylated by DNA methyltransferases Dnmt3A and Dnmt3B and methylation is maintained when new cells are formed by Dnmt1. But Dnm1 also needs to be regulated by Histone H3 ubiquitylation. DNA replication is done by Uhrf1 protein which is loaded onto chromatin so hemi-methylated DNA can be converted to fully-methylated DNA (Goll and Bestor 2005). Protein deubiquitylation is a very complicated process but deubiquitylating enzymes (DUBs) in USP7 regulates the stability of proteins (Du and Cheng 2010a, 2015b). In this study, it is shown that Usp7 is involved in the maintenance but also the depletion of DNA methylation in Xenopus and mammals.
This study showed when pan-DUB activity is inhibited by Ub-VS by suppressing protein ubiquitylation it blocked efficient DNA methylation but it has no effect on DNA replication in Xenopus egg extracts. Free ubiquitin was then added to the extracts treated with Ub-VS which restored ubiquitylation thus restoring DNA methylation (Dimova and Long 2012a, 2014b).
Xenopus egg extracts are prepared in the lab to investigate chromosomal replication. The Xenopus egg extracts were supplemented with an energy regeneration mix made of 2 mM ATP, 20 mM phosphocreatine, and 5 μg/ml creatine kinase (Yamaguchi et al. 2017). Some demembranated sperm nuclei were added to the egg extracts and incubated at 22 °C. Each replication was repeated three times. DNA replication was inhibited by incubating low-speed supernatants (LSS) with germin for about 10 minutes.
To deplete Usp7, 30 μg of anti-Usp7 antibodies were coupled with 40 μl of Protein A Sepharose overnight at 4 °C. The antibody product was then washed three times with 400 μl CPB (2% sucrose). A ratio of 1: 0.2 of LSS to antibody product was incubated for 1 hour at 4 °C.
Dnmt1 depletion resulted in an accumulation of ubiquitylated histone H3. Mass spectrometry analysis was done and it was found that Usp7 forms a complex with Dnmt1 in egg extracts and that was the reason for its accumulation. In addition, it was proven that Chromatin loading of Usp7 is dependent on its assembly with Dnmt1 and Uhrf1 for DNA replication. Furthermore, Usp7 regulated maintenance of DNA methylation and Usp7 depletion actually resulted in the accumulation of Dnmt1 in chromatin during DNA replication.
This Study was used to demonstrate that Usp7 is used in regulating the maintenance of DNA methylation through deubiquitylation of ubiquitylated histone H3 (Yamaguchi et al. 2017). Throughout this lab it was observed that Usp7 forms a stable complex with Dnmt1 in both Xenopus egg extracts and mammalian cells. Also, chromatin loading of Usp7 is dependent on ongoing DNA replication and requires both Dnmt1 and Uhrf1 in egg extracts. When Usp7 is depleted that results in a buildup of ubiquitylated histone H3 in both egg extracts and mammalian cells. Usp7 interacts with Dnmt1 and Uhrf1 by preventing their polyubiquitylation and proteasomal degradation (Du et al. 2010). Chromatin loading of Uhrf1 is enhanced by Usp7 depletion because of the suppression of histone H3 deubiquitylation and enhancement of fully-methylated DNA.
It was noticed that when Usp7 regulation is reduced in mammalian cells it results in a reduction in the level of DNA methylation. Also, the interaction between Dnmt1 and ubiquitylated histone reduces DNA methylation during DNA replication. Dnmt1 undergoes a conformational change in its catalytic pocket when it is involved with histone H3 which leads to its enzymatic activation but removing ubiquitin from histone H3 by Usp7 has shown to regulate Dnmt1’s catalytic activity. Therefore, Usp47 is not a major deubiquitylating enzyme that regulates recruitment of Dnmt1 to DNA replication sites. Studies by Cheng et al. (2015) supported this study’s findings that Dnmt1 depletion resulted in an accumulation of ubiquitylated histone H3.
Through evidence it was shown that to stabilize USP7, DNMT1 needs to be regulated by acetylation. To add on the study done by Du et al. (2010) supported the idea that DNMT1 is the main enzyme to control DNA methylation.
We provide you with original essay samples, perfect formatting and styling
To export a reference to this article please select a referencing style below:
Sorry, copying is not allowed on our website. If you’d like this or any other sample, we’ll happily email it to you.
Attention! This essay is not unique. You can get a 100% Plagiarism-FREE one in 30 sec
Sorry, we could not paraphrase this essay. Our professional writers can rewrite it and get you a unique paper.
Please check your inbox.
Want us to write one just for you? We can custom edit this essay into an original, 100% plagiarism free essay.Order now
Are you interested in getting a customized paper?Check it out!