AbstractIn nearly all eukaryotic cells, genomic DNA is condensed into the cell nucleus through association with histones to form a nucleoprotein complex called chromatin. The structure of chromatin affects all DNA-dependent processes, in particular transcription, replication and DNA repair. Histone variants and histone post-translational modifications can alter the structure of chromatin, which in turn governs access of regulatory factors to the DNA. Presented in this thesis is an investigation, using X. laevis, to study the functions of histone variants and two potential histone modifying proteins, hnRNP AB and hnRNP D, in the early vertebrate embryo.
Spatial and temporal mRNA expression analyses of the histone variants and hnRNPs were performed using wholemount in situ hybridisation (WISH), demonstrating various mRNA levels at differing points in the embryos. At the later stages of embryogenesis the majority of the transcripts were expressed in the anterior neural tissues, which correlated with cell proliferation. To investigate the roles of two recently identified isoforms of the histone variant H2A.Z and the two hnRNP proteins, loss of function studies were performed using morpholino oligonucleotides. Loss of both hnRNP AB and hnRNP D resulted in paralysis of the embryos. Phenotypic analysis of these morphant embryos revealed a decrease in the number of primary neurons, whilst hnRNP D morphants additionally lacked blood differentiation. Inhibition of H2A.Z2 expression likewise caused paralysis. In embryos lacking H2A.Z2 however, a significant increase in primary neurons was observed, although other developmental pathways were unaffected. This increase appeared to be caused by a down-regulation of Notch expression, which rescue experiments showed to be due to the specific loss of H2A.Z2. The loss of H2A.Z1 resulted in slowly developing embryos having morphological defects. Phenotypic analysis showed that the inhibition of H2A.Z1 caused problems in the development of many tissues. A down-regulation of Xbra in these H2A.Z1 morphant embryos resulted in a decrease in mesoderm induction, shown by rescue experiments to be due to the specific loss of H2A.Z1. The effect on the control of Xbra was direct since signalling and transduction functioned normally and acetylated H2A.Z1 was seen at the Xbra promoter by ChIP experiments.
The data presented here serve to further understanding of the gene regulatory roles of chromatin structure during development and shows that hnRNP AB, hnRNP D and the H2A.Z isoforms are crucial for normal embryogene
|Date of Award
|Matt Guille (Supervisor), Fiona Myers (Supervisor) & Tim Hebbes (Supervisor)