Inhaltszusammenfassung

DNA demethylation plays an important role in development and vertebrate physiology. In active DNA demethylation, 5-methylcytosine (5mC) is iteratively oxidized into 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-caboxylcytosine (5caC) by Ten-eleven-translocation (TET) enzymes. Subsequently, 5fC and 5caC are removed by Thymine DNA glycosylase (TDG) and base excision repair (BER) proteins. In vitro data indicate that Nei-like (NEIL) glycosylases play a crucial role in this contex...DNA demethylation plays an important role in development and vertebrate physiology. In active DNA demethylation, 5-methylcytosine (5mC) is iteratively oxidized into 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-caboxylcytosine (5caC) by Ten-eleven-translocation (TET) enzymes. Subsequently, 5fC and 5caC are removed by Thymine DNA glycosylase (TDG) and base excision repair (BER) proteins. In vitro data indicate that Nei-like (NEIL) glycosylases play a crucial role in this context by increasing the enzymatic turnover of TDG in BER. In the first part of my thesis, I analyzed the role of Tet3, Tdg, and especially Neil proteins in active DNA demethylation in Xenopus laevis embryos. Expression analysis showed that tet3, tdg, neil1 and neil3 are expressed during the embryonic development of the central nervous system. Neil2 mRNA is maternally supplied, and its expression level is decreased drastically upon zygotic genome activation (ZGA). Analysis of 5mC and its oxidative derivatives by mass spectrometry supports the cooperation of Tet3, Tdg, and Neil2 in active DNA demethylation in vivo. Antisense Morpholino (MO) knockdown of Tet3, Tdg, Neil2 and Neil3 induces neural crest defects and microcephaly. In the second part of my thesis, I examined the mechanisms leading to the neural crest cell (NCC) defects and microcephaly phenotypes of neil2 MO injected embryos (Neil2 morphants). Whole transcriptome analysis and qPCR expression analysis show that the Tp53 DNA damage pathway is activated in Neil2 morphants. In Neil2 morphant neural plates, pS345 Chk1, Tp53, and active caspase-3 accumulate during NCC differentiation. Knockdown of Tp53 reduces apoptotic cell death and rescues microcephaly in Neil2 morphants. Neural crest defects and microcephaly are recapitulated by treating embryos with the reactive oxygen species (ROS) inducer pyocyanin. This treatment upregulates protein levels of pS345 Chk1, Tp53, and active caspase-3, and activates expression of Tp53 target genes. Pyocyanin and neil2 MO injection show synergistic effects in inducing microcephaly. Furthermore, Neil2 morphants display craniofacial abnormalities, mimicking Treacher-Collins-Syndrome. In summary, my study demonstrates a novel role of Neil2 in cooperation with Tet3 and Tdg in active DNA demethylation during Xenopus embryogenesis. In addition, Neil2 deficiency elicits an oxidative stress-induced, Tp53-dependent DNA damage response, which impairs NCC differentiation. My work emphasizes how defects in BER can lead to a selective lineage defect during embryogenesis.» weiterlesen» einklappen

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DDC Sachgruppe:
Biowissenschaften, Biologie