Sortase-mediated ubiquitylation of Histone H2B and its biological consequences

Regulation of gene expression has been a longstanding puzzle in the understanding of cell biological processes. Histone 2B (H2B) ubiquitylation has been suggested to collaborate in the complex mechanisms that control the activation or silencing of genes. Here we try to explain how we could use the sortagging technique to control ubiquitylation events inside the nuclear envelope. We were able to perform in Saccharomyces cerevisiae an in vivo SrtA-mediated intramolecular circularization reaction of the NLS-tagged enhanced green fluorescent protein that contains an Nterminal glycine residue and a C-terminal sortagging motif (G-NLS-eGFP-LPETGmyc). However, nuclear fractionation experiments were unable to show efficient nuclear localization of NLS-SrtA and the modified eGFP. We designed and expressed in yeast a sortaggable H2B molecule that could be used for intranuclear sortase-mediated histone ubiquitylation. For the benefit of future SrtA experiments, we produced mouse polyclonal antibodies against Staphylococcus aureus and Streptomyces pyogenes SrtA. These experiments will help to further the development of intranuclear sortagging reactions in yeast and to apply the technique to perform inducible H2B ubiquitylation.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2013

Author dc:creator

• Díaz-Torres, José J. (José Javier)

Advisor dc:contributor.advisor

• Hidde L. Ploegh.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/79180

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/79180