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Characterization of histone acetyltransferase KAT6A and transcriptional repressor SAMD1 – two proteins possessing a novel class of unmethylated DNA-binding winged helix domains
This thesis addresses the functions of two transcriptional regulators; histone acetyltransferase and transcriptional activator KAT6A (MOZ), and transcriptional repressor SAMD1 (Atherin), in their respective molecular and cell type-specific roles. Firstly, we identified a novel winged helix domain a...
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| Format: | Dissertation |
| Langue: | anglais |
| Publié: |
Philipps-Universität Marburg
2023
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| Accès en ligne: | Texte intégral en PDF |
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| Résumé: | This thesis addresses the functions of two transcriptional regulators; histone acetyltransferase and transcriptional activator KAT6A (MOZ), and transcriptional repressor SAMD1 (Atherin), in their respective molecular and cell type-specific roles.
Firstly, we identified a novel winged helix domain at the N-terminal end of KAT6A as a distinct feature of the previously described NEMM (N-terminal part of Enok, MOZ or MORF) domain, a homologous region with other histone acetyltransferases. This KAT6A WH1 domain possesses a DNA-recognition motif, directly recruiting KAT6A to unmethylated CpG-rich DNA. Analyses of genome-wide ectopic KAT6A binding sites in HEK293 cells revealed a high correlation with unmethylated CpG islands (CGIs), particularly with CGIs associated with active promoters, decorated with the histone marks H3K4me3 and H3K9ac. We demonstrated that KAT6A WH1 is necessary for the observed recruitment to CGIs, but by itself not sufficient and dependent on KAT6A WH2 and DPF domains for effective KAT6A-CGI interactions. Indeed, mutations of the KAT6A WH1 domain caused a complete abrogation of KAT6A binding to CGIs, but increased the binding to gene body regions at a subset of target genes, indicating that recruitment mechanisms to these sites are independent of KAT6A WH1. This study, for the first time, demonstrates a direct chromatin recruitment mechanism of a histone acetyltransferase. Moreover, it provides new and more detailed insights into KAT6A chromatin recognition and target site association to facilitate histone acetylation and enforce transcription control.
Secondly, we morphologically characterized homozygous and heterozygous SAMD1 knockout (KO) mouse embryos and their respective phenotypes in embryogenesis. The homozygous deletion of SAMD1 was embryonic lethal, while heterozygous SAMD1 KO mice were born alive. At embryonic day E14.5 severe degradation of internal organs and formation of subcutaneous oedema were visible in SAMD1 KO embryos, most likely due to disturbed blood vessel maturation and resulting hypoxia. The observed defects led to the conclusion that SAMD1 is required for functional angiogenesis and the development of the cardiovascular system. Furthermore, craniofacial defects upon SAMD1 KO indicated malfunctions in head and brain development. To gain further insight into the role of SAMD1 during neurogenesis, we established a directed neural differentiation approach of murine embryonic stem cells (mESCs). Transcriptional dysregulation and the global increase of H3K4me2 were observed in SAMD1 KO cells during this process, further supporting a functional role of SAMD1 in neural cell development.
Overall, this study sheds light on the underlying molecular mechanism of KAT6A binding to chromatin and emphasizes the similarities of KAT6A WH1 to the previously described homologous SAMD1 WH domain, which directly interacts with unmethylated CGIs and facilitates transcription regulation, as well. Both KAT6A and SAMD1 are involved in a variety of developmental processes and dysregulations are associated with cellular abnormalities and the onset of cancer. |
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| DOI: | 10.17192/z2023.0558 |