Characterisation of AEBP2: a polycomb repressive complex 2 component
<p>Polycomb complexes act as repressors of transcription at many developmentally important genes. Repression by polycomb-repressive complex 2 (PRC2) is mediated by the deposition of a tri-methylation mark on lysine 27 of the histone H3 tail (H3K27me3). The PRC2 complex consists of three core p...
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| Format: | Thesis |
| Language: | English |
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2013
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| _version_ | 1826315725668614144 |
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| author | Grijzenhout, A |
| author2 | Brockdorff, N |
| author_facet | Brockdorff, N Grijzenhout, A |
| author_sort | Grijzenhout, A |
| collection | OXFORD |
| description | <p>Polycomb complexes act as repressors of transcription at many developmentally important genes. Repression by polycomb-repressive complex 2 (PRC2) is mediated by the deposition of a tri-methylation mark on lysine 27 of the histone H3 tail (H3K27me3). The PRC2 complex consists of three core proteins: EED, SUZ12, and one of either EZH2 or EZH1, which contain the enzymatic activity. The zinc finger containing protein AEBP2 (AE1 binding protein 2) has been co-purified with the PRC2 complex. The aim of this thesis was to study the function of AEBP2. Chromatin immunoprecipitation experiments indicate that AEBP2 is enriched at the same genomic locations as H3K27me3 genomewide. Using an X inactivation model, I found that AEBP2 and H3K27me3 also co-localise by immunofluorescence upon <em>Xist</em> induction.</p> <p>Large scale immunoprecipitation followed by mass spectrometry confirmed the interaction of AEBP2 with PRC2 complex members. Interestingly, I did not detect any peptides of the Polycomblike (PCL) family proteins. These proteins have previously been shown to interact with the PRC2 complex. Their absence in the AEBP2 immunoprecipitation, together with published work demonstrating an absence of AEBP2 in PCL purifications, suggests that AEBP2 and PCL family proteins might be mutually exclusive in PRC2 complexes.</p> <p>I have created an embryonic stem cell line in which both alleles of <em>Aebp2</em> contain a gene-trap. This cell line was used to address the suggestion that AEBP2 might use its zinc finger domain to act as a sequence-specific PRC2 recruitment factor. AEBP2-depleted cells do not show diminished recruitment of PRC2. Additionally, electromobility shift assays failed to show that AEBP2 could bind DNA. Furthermore, recruitment of AEBP2 to genomic targets depended on the presence of the core PRC2 protein EED. Together, this makes it unlikely that AEBP2 acts as a sequence-specific PRC2 recruitment factor.</p> <p>In keeping with the lack of effect on PRC2 enrichment, preliminary experiments have shown that AEBP2 depletion does not greatly affect levels of H3K27me3, or the expression of PRC2 target genes. The <em>Aebp2</em> gene-trapped cell line will be used to investigate further functions of AEBP2. Additionally, these cells have been used for blastocyst injection to create an <em>Aebp2</em> gene-trapped mouse. Future work will show the effect of AEBP2 depletion on development.</p> |
| first_indexed | 2024-03-06T19:07:19Z |
| format | Thesis |
| id | oxford-uuid:159716a1-a03c-44f3-9fd1-0e88328caef6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:31:17Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:159716a1-a03c-44f3-9fd1-0e88328caef62024-12-01T14:39:11ZCharacterisation of AEBP2: a polycomb repressive complex 2 componentThesishttp://purl.org/coar/resource_type/c_db06uuid:159716a1-a03c-44f3-9fd1-0e88328caef6Cell Biology (see also Plant sciences)BiologyEnglishOxford University Research Archive - Valet2013Grijzenhout, ABrockdorff, N<p>Polycomb complexes act as repressors of transcription at many developmentally important genes. Repression by polycomb-repressive complex 2 (PRC2) is mediated by the deposition of a tri-methylation mark on lysine 27 of the histone H3 tail (H3K27me3). The PRC2 complex consists of three core proteins: EED, SUZ12, and one of either EZH2 or EZH1, which contain the enzymatic activity. The zinc finger containing protein AEBP2 (AE1 binding protein 2) has been co-purified with the PRC2 complex. The aim of this thesis was to study the function of AEBP2. Chromatin immunoprecipitation experiments indicate that AEBP2 is enriched at the same genomic locations as H3K27me3 genomewide. Using an X inactivation model, I found that AEBP2 and H3K27me3 also co-localise by immunofluorescence upon <em>Xist</em> induction.</p> <p>Large scale immunoprecipitation followed by mass spectrometry confirmed the interaction of AEBP2 with PRC2 complex members. Interestingly, I did not detect any peptides of the Polycomblike (PCL) family proteins. These proteins have previously been shown to interact with the PRC2 complex. Their absence in the AEBP2 immunoprecipitation, together with published work demonstrating an absence of AEBP2 in PCL purifications, suggests that AEBP2 and PCL family proteins might be mutually exclusive in PRC2 complexes.</p> <p>I have created an embryonic stem cell line in which both alleles of <em>Aebp2</em> contain a gene-trap. This cell line was used to address the suggestion that AEBP2 might use its zinc finger domain to act as a sequence-specific PRC2 recruitment factor. AEBP2-depleted cells do not show diminished recruitment of PRC2. Additionally, electromobility shift assays failed to show that AEBP2 could bind DNA. Furthermore, recruitment of AEBP2 to genomic targets depended on the presence of the core PRC2 protein EED. Together, this makes it unlikely that AEBP2 acts as a sequence-specific PRC2 recruitment factor.</p> <p>In keeping with the lack of effect on PRC2 enrichment, preliminary experiments have shown that AEBP2 depletion does not greatly affect levels of H3K27me3, or the expression of PRC2 target genes. The <em>Aebp2</em> gene-trapped cell line will be used to investigate further functions of AEBP2. Additionally, these cells have been used for blastocyst injection to create an <em>Aebp2</em> gene-trapped mouse. Future work will show the effect of AEBP2 depletion on development.</p> |
| spellingShingle | Cell Biology (see also Plant sciences) Biology Grijzenhout, A Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title | Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title_full | Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title_fullStr | Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title_full_unstemmed | Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title_short | Characterisation of AEBP2: a polycomb repressive complex 2 component |
| title_sort | characterisation of aebp2 a polycomb repressive complex 2 component |
| topic | Cell Biology (see also Plant sciences) Biology |
| work_keys_str_mv | AT grijzenhouta characterisationofaebp2apolycombrepressivecomplex2component |