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Title: Identification and characterisation of alternative forms of SETD2/HYPB (SET domain-containing protein 2 / Huntingtin yeast partner B)
Author: Lee, Benjamin Mark
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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SETD2/HYPB (SET domain-containing protein 2 / Huntingtin yeast partner B) is the predominant lysine methyltransferase in mammals that mediates histone H3 lysine-36 (H3K36) trimethylation, which is associated with transcription elongation and RNA splicing. SETD2 is further implicated in p53 function, vascular development, cancer progression and, through Huntingtin-interaction, Huntington's disease. Although different transcripts and putative protein isoforms have been detected previously, their identity, function and significance have not been rigorously investigated. This thesis aims to identify and characterise endogenous transcripts and protein isoforms of SETD2 in mouse fibroblasts. Affnity-purified N- and C-terminal antibodies specifically detected the ≈ 290 kDa methyltransferase (p290SETD2), verified by RNAi, in addition to N terminal-specific ≈ 120 kDa protein, and C terminal-specific forms at ≈ 140 and ≈ 66 kDa (p66), which all appeared too stable to deplete by transient siRNA transfection. Conserved in human and mouse cells, immunodetection of p66 exhibited unusual requirement for denaturation with urea at 95°C. Subcellular fractionation revealed distinct extraction properties of putative isoforms and facilitated partial purification of p66 for proteomic analysis. Co-fractionation and co migration by two-dimensional gel electrophoresis of p66 detected by two independent C terminal antibodies suggested it represents a novel C terminal-specific isoform. Reverse transcription−PCR and DNA-sequencing demonstrated the existence of multiple, alternatively-spliced Setd2 transcripts that plausibly generate truncated proteins. A transcript variant containing a novel complete open-reading-frame, consistent for p66 generation, was identified. Its ectopic expression in mouse fibroblasts produced a distinct SETD2 isoform, whose physical and extraction characteristics were studied in comparison with endogenous immunoforms. In summary, this thesis demonstrates that multiple alternatively-spliced transcripts arise from the Setd2 gene, consistent with immunodetection of several C- and N-terminal-specific putative SETD2 isoforms, additional to the H3K36 methyltransferase. Verification of these isoforms by independent methods would have implications for proposed interactions and function of SETD2 in transcription, epigenetics, cancer development and Huntington’s disease.
Supervisor: Mahadevan, Louis C.; Mancini, Erika J. Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; Biology ; histone ; nucleosome ; chromatin ; methylation ; methyltransferase ; H3K36me3 ; KMT3a ; isoform ; denaturation ; urea