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Investigation of the epigenetic protein landscape using proteomics-based strategies
Investigation of the epigenetic protein landscape using proteomics-based strategies
Epigenetic gene regulation predominantly depends on proteins which modify histones, remodel chromatin structure and set or remove DNA methylation marks. In this study, we investigated the epigenetic protein landscape on different functional levels using mass spectrometry-based approaches. First, the local protein environment at a given DNA sequence can dramatically differ depending on the chromatin type, e.g. at euchromatic or heterochromatic regions. We developed a new strategy to investigate such local chromatin environments. By combining the programmable DNA binding of an inactive dCas9 protein with a promiscuous biotin ligase (BirA*), the heterochromatic DNA sequences of telomeres, major satellites and minor satellites were targeted and proteins binding to those regions were selectively labeled with biotin which enabled enrichment and protein identification via mass spectrometry. Using this CasID strategy, we found a novel candidate protein, ZNF512 (zinc finger protein 512), to be localized at heterochromatic regions. Second, we investigated epigenetic protein complex associations of the methylcytosine oxidase TET1 in mouse embryonic stem cells as well as in in vitro differentiated epiblast-like cells. For this purpose, a novel genome engineering strategy, termed MIN-tag technique was used to insert functional cassettes into the endogenous Tet1 locus. We performed GFP-pulldown experiments followed by mass spectrometry as well as proximity-dependent protein identification (BioID) and found that in case of the big, presumably unstructured and tightly chromatin associated protein TET1, BioID is favourable over affinity purification approaches to capture known and novel interacting proteins. The obtained dataset draws a complex picture of TET1-containing complexes with involvement in transcriptional regulation and chromatin remodeling. Importantly, we identified several novel putative interactors of TET1, e.g. the glutamine and serine rich protein QSER1. Finally, on the single protein level, post-translational modifications such as ubiquitination can significantly affect protein function. Here, the ubiquitination activity of the E3-ligase proteins and epigenetic regulators UHRF1 and UHRF2 was investigated. To this end, we performed a mass spectrometry-based screen for potential UHRF ubiquitination targets in mouse embryonic stem cells depleted for UHRF1 and UHRF2. Among numerous known and novel identified ubiquitination targets, we found PCNA-associated factor 15 (PAF15) ubiquitination to be dependent on UHRF1.
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Karg, Elisabeth
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Karg, Elisabeth (2018): Investigation of the epigenetic protein landscape using proteomics-based strategies. Dissertation, LMU München: Faculty of Biology
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Abstract

Epigenetic gene regulation predominantly depends on proteins which modify histones, remodel chromatin structure and set or remove DNA methylation marks. In this study, we investigated the epigenetic protein landscape on different functional levels using mass spectrometry-based approaches. First, the local protein environment at a given DNA sequence can dramatically differ depending on the chromatin type, e.g. at euchromatic or heterochromatic regions. We developed a new strategy to investigate such local chromatin environments. By combining the programmable DNA binding of an inactive dCas9 protein with a promiscuous biotin ligase (BirA*), the heterochromatic DNA sequences of telomeres, major satellites and minor satellites were targeted and proteins binding to those regions were selectively labeled with biotin which enabled enrichment and protein identification via mass spectrometry. Using this CasID strategy, we found a novel candidate protein, ZNF512 (zinc finger protein 512), to be localized at heterochromatic regions. Second, we investigated epigenetic protein complex associations of the methylcytosine oxidase TET1 in mouse embryonic stem cells as well as in in vitro differentiated epiblast-like cells. For this purpose, a novel genome engineering strategy, termed MIN-tag technique was used to insert functional cassettes into the endogenous Tet1 locus. We performed GFP-pulldown experiments followed by mass spectrometry as well as proximity-dependent protein identification (BioID) and found that in case of the big, presumably unstructured and tightly chromatin associated protein TET1, BioID is favourable over affinity purification approaches to capture known and novel interacting proteins. The obtained dataset draws a complex picture of TET1-containing complexes with involvement in transcriptional regulation and chromatin remodeling. Importantly, we identified several novel putative interactors of TET1, e.g. the glutamine and serine rich protein QSER1. Finally, on the single protein level, post-translational modifications such as ubiquitination can significantly affect protein function. Here, the ubiquitination activity of the E3-ligase proteins and epigenetic regulators UHRF1 and UHRF2 was investigated. To this end, we performed a mass spectrometry-based screen for potential UHRF ubiquitination targets in mouse embryonic stem cells depleted for UHRF1 and UHRF2. Among numerous known and novel identified ubiquitination targets, we found PCNA-associated factor 15 (PAF15) ubiquitination to be dependent on UHRF1.