Home Page 1

Reference URL

Comment

Save to favorites

To link to the entire object, paste this link in email, IM or document

To embed the entire object, paste this HTML in website

To link to this page, paste this link in email, IM or document

To embed this page, paste this HTML in website

Identification and characterization of PR-Set7 and histone H4 lysine 20 methylation-associated proteins

Page 1

Save page

1 of 153

View Description

View PDF & Text

Download

small (250x250 max)

medium (500x500 max)

large ( > 500x500)

Full Resolution

All (PDF)

This page

All

Subset

PDF
Text

Text Search...

Object Description

Title	Identification and characterization of PR-Set7 and histone H4 lysine 20 methylation-associated proteins
Author	Spektor, Tanya Magazinnik
Author email	magazinn@usc.edu; tanyamspektor@gmail.com
Degree	Doctor of Philosophy
Document type	Dissertation
Degree program	Biochemistry & Molecular Biology
School	Keck School of Medicine
Date defended/completed	2009-08-27
Date submitted	2009
Restricted until	Unrestricted
Date published	2009-11-18
Advisor (committee chair)	Rice, Judd C.
Advisor (committee member)	Laird, Ite Stallcup, Michael Coetzee, Gerry
Abstract	Chromatin is the complex of DNA, histones, and nonhistone proteins found in the nucleus of a eukaryotic cell and is known to play a critical role in regulation of DNA-templated processes such as gene transcription, DNA replication, and DNA repair. The numerous potential covalent modifications of the histone proteins help direct the recruitment of specific nuclear factors to these modifications thus causing changes in biological outcomes, phenomenon also known as “histone code”. One example of such modification is the addition of methyl groups to lysine residues on N-terminal histone tails by enzymes known as histone methyltransferases (HMTases). We previously identified that PR-Set7 specifically methylates Lysine 20 of Histone H4 (H4K20); a mark associated with transcriptional repression, condensed chromatin, and DNA repair. Our biochemical purification of the native complex indicates that it is a part of a large multi-protein complex. We hypothesize that these unidentified proteins play a critical role in regulation of PR-Set7 function and H4K20me1 mediated pathways. In this study we are using immunoaffinity purification and yeast two-hybrid techniques to identify PR-Set7-associated proteins and a novel Mammalian Tethered Catalysis (MTeC) method to identify H4K20 methyl binding proteins.; A tandem tag immunoaffinity purification (IAP) using full length PR-Set7 revealed that components of the DNA damage machinery, such as Ku70/80, DNA-PK and PARP1, are part of its multi-protein complex. These findings provided evidence for a novel function of PR-Set7 in the DNA repair pathway.; In a parallel approach, yeast two-hybrid assay was performed using full length PR-Set7 as “bait”. Yeast two-hybrid screen revealed a novel interaction between PR-Set7 and the sumoylation-conjugation enzyme, UBC9. Additional studies demonstrated that direct interaction with UBC9 is mediated by the N-terminal (non-catalytic) domain of PR-Set7. Furthermore, we discovered that PR-Set7 is covalently modified specifically with SUMO-1 and that this occurs in an ARIP3, E3 ligase-dependent manner in vivo. Importantly, depletion of UBC9 in cells resulted in the derepression of PR-Set7 target genes strongly suggesting that sumoylation by UBC9 is required for the normal repressive effects of the PR-Set7 H4K20 monomethyltransferase.; Lastly, we developed and validated an innovative in vivo approach called Mammalian Tethered Catalysis (MTeC). Using methylated histones and methyl-specific histone binding proteins as the proof-of-principle, we determined that the simple MTeC approach can compliment existing in vitro binding methods and can also provide unique in vivo insights into PTM-dependent interactions. For example, we confirmed previous in vitro findings that endogenous HP1 preferentially binds H3K9me3. However, in contrast to recent in vitro observations, MTeC revealed that the tandem tudor domain-containing proteins, JMJD2A and 53BP1, display no preferential H4K20 methyl-selectivity in vivo. Finally, using MTeC in an unbiased manner we discovered novel H3K9 and H4K20 methyl-specific PTMBPs. First, we determined that endogenous G9a binds methylated H3K9 in vivo. Further use of MTeC to characterize this interaction revealed that G9a selectively binds H3K9me1 in vivo but not H3K9me2, contrary to recent in vitro findings. Second, we discovered and confirmed that Ku70 is a novel H4K20me1 binding protein.; In summary, we have provided evidence supporting a novel role of PR-Set7 and H4K20me1 in protecting specific genomic regions from the DNA damage. Based on the fact that PR-Set7 interacts with the components of the DNA damage machinery (Ku70/80, DNA-PK and PARP1) and that H4K20me1 itself recruits Ku70 component of Ku70/80 heterodimer, we hypothesize that PR-Set7 is targeted to the DNA damage sensitive regions of the genome to provide direct layer of protection and prevent accumulation of the DNA breaks; deregulation of PR-Set7 mediated pathway will lead to cumulative DNA damage and genomic instability, two key events associated with oncogenesis.
Keyword	chromatin; epigenetics; histone methylation; histone H4 lysine 20; sumoylation; DNA repair; Ku70/80; PARP1; PR-Set7; UBC9; mammalian tethered-catalysis (MTeC); methyl-binding proteins
Language	English
Part of collection	University of Southern California dissertations and theses
Publisher (of the original version)	University of Southern California
Place of publication (of the original version)	Los Angeles, California
Publisher (of the digital version)	University of Southern California. Libraries
Provenance	Electronically uploaded by the author
Type	texts
Legacy record ID	usctheses-m2746
Rights	Spektor, Tanya Magazinnik
Repository name	Libraries, University of Southern California
Repository address	Los Angeles, California
Repository email	http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/
Filename	etd-Spektor-3304
Archival file	uscthesesreloadpub_Volume56/etd-Spektor-3304.pdf