Beta-Globin Gene Regulation and Nuclear Organisation
All genetic information required for the development and functioning of an organism is stored in billions of base pairs of deoxyribonucleic acid (or DNA). In eukaryotes, DNA is organised in large units called chromosomes that are located inside the cells nucleus. On these chromosomes reside functional units called genes, which encode for discrete hereditary characteristics, in most cases proteins. Multicellular organisms are composed of numerous different cell types, such as blood, skin, muscle and brain cells. This cellular diversity is extraordinary given the fact that all of these individual cell types harbour the same genetic material. The functional and morphological diversity among cells is dictated by the precise regulation of genes throughout development and different stages of cellular differentiation. Incorrect spatiotemporal activation and repression of genes can lead to cellular defects, which may eventually result in diseases such as cancer. Th! erefore, different mechanisms of control exist that regulate the transcription of genes in time and space, and understanding this transcriptional regulation in more detail is one of the key objectives in the field of molecular biology. Different cis-regulatory DNA elements like promoters, enhancers and insulators are involved in the regulated expression of protein coding genes throughout development. An important question is how these distal cis-regulatory DNA elements communicate over distance to activate tissue- and developmental stage-specific gene expression. In this thesis, the mouse Î²-globin locus is used as a model system to study transcriptional regulation in the context of the living nucleus. The genes of this locus all encode for the Î²-globin like protein that together with Î±-globin and heme forms a functional hemoglobin molecule, which is capable of binding oxygen and carbon dioxide in a reversible manner in red blood cells (erythrocytes). Besides the Î²-globin genes, the locus contains a number of well-characterised cis-regulatory DNA elements like enhancers, promoters and a locus control region (LCR). Previously, the structural organisation of the Î²-globin locus in its native context was s! tudied by applying Chromosome Conformation Capture (3C) technology. These experiments showed that the locus adopts a red blood cell specific organisation in which cis regulatory elements of the LCR and the active Î²-globin genes spatially cluster. In addition to LCR-gene interactions these studies revealed erythroid specific long-range interactions with other sets of hypersensitive sites residing upstream and downstream of the Î²-globin locus. Collectively this spatial conformation of the Î²-globin locus was named the Active Chromatin Hub (ACH). However, little is known about the molecular events that accompany and underlie ACH formation in the Î²-globin locus during erythroid differentiation. Chapter 3 of this thesis describes experiments in which recently established I/11 erythroid progenitor cells are used as a model system to study the molecular events involved in the establishment of contacts between the LCR and the Î²-globin genes. The results show that upon induction of I/11 cells, the Î²-globin like genes are transcribed at rates similar to those observed in vivo and differentiation is accompanied by an increased ratio of positive versus negative chromatin modifications at the Î²major promoter and the HSs of the LCR. In addition, the data show that binding of the erythroid-specific transcription factors GATA-1 and EKLF to the locus, while previously shown to be required, is not sufficient for ACH formation. Moreover, it was demonstrated that in p45 NF-E2 knockout mice long-range contacts in the Î²-globin locus are formed normally, indicating that the erythroid-specific transcription factor p45 NF-E2 is dispensable for ACH formation. The role of the vertebrate insulator protein CTCF in long-range interactions in the Î²-globin locus is addressed in Chapter 4. CTCF binding sites flank the Î²-globin locus and have previously been shown to participate in erythroid specific spatial interactions in the context of the ACH. After conditional deletion of CTCF and targeted disruption of the 3â?THS1 CTCF-binding site, long-range interactions in the Î²-globin locus were destabilised. However, Î²-globin gene transcription was not measurably affected by the loss of these interactions and disruption of CTCF binding only caused local loss of histone acetylation and gain of histone methylation. This data demonstrates that CTCF is directly involved in long-range chromatin looping in the Î²-globin locus and regulates the local balance between active and repressive chromatin marks. 3C technology enables researchers to study the structural organisation of individual loci at high resolution. Recently, a number of 3C based strategies have been developed that allow screening of the entire genome in an unbiased manner for DNA segments that physically interact with a DNA fragment of choice. Application of these new methods is expected to provide exciting new insights into the conformational structure of selected chromosomal regions in the genome. However, 3C and 4C based methods also have important limitations and preconditions that need to be recognised and addressed properly. Chapter 2 of this thesis therefore describes and evaluates the most commonly used 3C-based methods and addresses potentials and pitfalls of these technologies. In Chapter 5 recently developed 4C technology was applied to study the nuclear organisation of both peri-centromeric and peri-telomeric chromosomal regions. The data show that there is a strong correlation between the transcriptional status of the selected peri-centromeric and peritelomeric regions and their interacting loci in the interphase nucleus. Moreover, interchromosomal interactions with peri-centromeric regions were biased towards other peri-centromeric regions and also peri-telomeric regions showed a preference to interact in trans with other peri-telomeric regions. The results demonstrate that both the transcriptional status of a genomic region and its proximity to defined repetitive sequences on the linear chromosome template strongly influence the positioning of a locus in the nucleus.
|Keywords||3C, 4C, Beta-globin, CTCF, NF-E2, chromatin, transcription|
|Promotor||F.G. Grosveld (Frank)|
|Publisher||Erasmus University Rotterdam|
|Sponsor||J.E. Jurriaanse Stichting|
Kooren, J.A.. (2007, December 19). Beta-Globin Gene Regulation and Nuclear Organisation. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/18433