One of the most important outstanding questions in biology involves the precise spatial and temporal regulation of gene activity, which enables different cell types to express the specific set of genes required for their function and is therefore a cornerstone for complex biological life. Cis-regulatory elements, such as gene promoters and enhancers, play a key role in controlling gene activity. These elements physically interact with the genes they regulate within structural chromatin domains. The organisation of chromosomes into these domains is critical for specific regulation of gene expression and disruption of these structures underlies common human disease. However, it is not understood how chromatin domains form, how interactions between the cis-regulatory elements contained within them are established, or how such interactions influence gene expression. The major hurdles in addressing these questions are to determine chromatin structures with high resolution and sensitivity and to examine their dynamic nature within single cells. To overcome these, I have developed Tri-C, a new chromosome conformation capture assay that can analyse concurrent chromatin interactions at single alleles at high resolution. By combining Tri-C with conventional chromosome conformation capture techniques, I have analysed the three-dimensional regulatory landscapes of the well-characterised murine globin loci at unprecedented depth. Additionally, to examine the roles of cis-regulatory elements in establishing chromatin architecture, I have analysed how engineered deletions in enhancers and CTCF-binding elements in the murine alpha-globin locus disrupt its chromatin landscape. These analyses reveal that the chromatin domains containing the globin genes represent compartmentalised structures, which are delimited by CTCF boundaries. The heterogeneity of interactions in these domains between individual cells is indicative for a dynamic process of loop extrusion underlying their formation. Within chromatin domains, preferential structures are formed in which multiple enhancers and promoters interact simultaneously. These complexes provide a structural basis for understanding how multiple cis-regulatory elements cooperate to establish robust regulation of gene expression. Importantly, these complex, tissue-specific structures, cannot be explained by loop extrusion alone and indicate other, independent mechanisms contributing to chromosome architecture, likely involving interactions mediated by multi-protein complexes. Together, these analyses show that the current view of genome organisation, in which chromosomes are organised by stable loops and domains that self-assemble into hierarchical structures, is not correct. Rather, chromatin architecture reflects a complex interplay between distinct molecular mechanisms contributing to the formation of regulatory landscapes that facilitate precise, robust control of gene expression.

Bibliography: 31 unnumbered leaves at end of vol

Typescript (photocopy)

Hemoglobin switching is a developmental process involving the dynamic transcriptional regulation of multiple globin genes. This molecular process involves multiple layer of complexity, and elucidating new mechanisms in this process will result in a more complete understanding of general gene regulation and will likely have direct clinical implications for hemoglobinopathies, such as sickle cell anemia. In this dissertation, I develop and characterize a new model for hemoglobin switching, the zebrafish. I defined and fully annotated the two zebrafish globin loci, termed major and minor loci. Both loci contain α– and β–genes oriented in a head–to–head fashion. Characterization of the globin expression pattern precisely defined the timing of normal switching and demonstrated that zebrafish, like humans, have two globin switches. The locus control region for the major locus was identified and in conjunction with a proximal promoter was able to generate robust, erythroid–specific expression in a transgenic line.

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Typescript (photocopy) / vii, 98 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1977

Thesis (Ph. D.)--University of Hong Kong, 1993.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Krüppel like factors (KLFs) are a family of 17 proteins whose main function is gene regulation by binding to DNA elements in the promoters of various genes. KLF transcription factors recognize CACCC-elements and act as activators or repressors of the gene expression. Among the 17 family members, KLF1, KLF2, and KLF4 share high homology to each other. KLF1 is the founding member of the family and is an erythroid-specific protein. KLF2 is expressed in erythroid, endothelial, and other cells. KLF4 is expressed in endothelial, smooth muscle, and other cells. In this thesis, the functions of these KLFs were reviewed in the context of subjects related to erythropoiesis and cardiovascular development. A mouse model lacking KLF1, KLF2, and KLF4 was used to investigate whether these genes have overlapping functions in regulating the embryonic β-globin genes during early embryogenesis. Quantitative RT-PCR assays were used to measure the expression level of Ey- and βh1- globin mRNA at embryonic day 9.5 (E9.5). It was found that KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos express significantly decreased amounts of Ey- and βh1-globin genes when compared to WT and KLF4-/- embryos. There were no significant changes in the levels of Ey- and βh1-globin mRNA between KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos. It was demonstrated here that KLF1 does not regulate KLF2 in mouse erythroid cells at E10.5.