In 1871, a unique substance was isolated from the white blood cells of pus. This substance, which later became known as chromatin, was shown to be a nucleoprotein complex which encompasses the majority of genomic DNA in all eukaryotes. Although chromatin was once viewed as primarily a structural component of the nucleus, it is now accepted that it also plays an important role in the modulation of transcription of individual genes. In this study, the 5S rRNA genes in Xenopus laevis were used as a system to investigate potential roles for chromatin structures in transcription regulation. X. laevis produces two major classes of 5S rRNA: the somatic type is present in most cells whereas the oocyte type is produced only during oogenesis and the early stages of embryogenesis. These two gene families share a very similar coding region and employ identical transcription machinery, leading researchers to believe that it is how these genes are packed into chromatin which is responsible for the differential developmental regulation.
Initially, this study focused on the binding constraints placed on the RNA polymerase III basal transcription factor, transcription factor IIIA (TFIIIA), by a histone octamer. Five overlapping fragments of the X. laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes and it was shown that each fragment positions a histone octamer at unique translational sites. Using these nucleosomes it was demonstrated that nucleosome translational positioning is the major determinant of the binding of TFIIIA to the 5S rRNA genes.
The relationship between core histone acetylation and transcription of the X. laevis 5S rRNA genes was also investigated. By immunopreciptitating chromatin fragments from a X. laevis kidney cell line with an antibody specific for hyperacetylated histone H4, it was shown that the oocyte 5S rRNA genes are packaged with hypoacetylated histone H4 when transcriptionally repressed.This taken together with the results of others, suggests a link between histone acetylation and RNA polymerase III transcription. However this study
was unable to shed light on the basis for this relationship as it was found that histone acetylation did not affect the binding of TFIIIA to nucleosomal DNA.
In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. It was shown that despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin and TFIIIA from Xenopus laevis.
Finally, it has long been proposed that selective binding of histone H1 is, in part, responsible for the differential developmental regulation of the oocyte and somatic 5S rRNA genes in Xenopus laevis. In this study it was shown that histone H1 bound both oocyte and somatic genes equally after reconstitution into mononucleosomes or oligonucleosome arrays. Furthermore it was shown that the binding of histone H1 selectively repressed only oocyte gene transcription, and that a RNA polymerase III selectively repressed only oocyte gene transcription, and that a RNA polymerase III transcription complex was able to initiate transcription of nucleosomal somatic templates regardless of whether histone H1 was present. These results support a model in which the differential regulation of the 5S rRNA genes is not due simply to the prevention of histone HI binding by transcription complexes on the somatic genes, but rather a difference in the interaction of histone HI with the somatic and oocyte genes. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8294 |
| Date | 22 June 2017 |
| Creators | Howe, LeAnn Judith |
| Contributors | Ausio, Juan |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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