CTCF: Comprehending The Complex Functions of an 11 zinc finger transcription factor

In a multi-cellular organism, every somatic cell nucleus broadly contains the same sequence of DNA, yet clearly most cells are very different to each other. Specific sets of genes encoding proteins become activated whereas others are repressed. Within the genome, independently regulated genes are often found in close proximity to other genes that have different patterns of expression. How specific gene loci are organised in nuclear space is only recently emerging. CTCF is a protein that has been strongly implicated in mediating many distinct processes of gene regulation, including transcription, chromatin structure, and the structural organisation of gene loci. The aim of this thesis was to investigate the function of the CTCF protein in vivo, in particular the role of CTCF in regulating cellular proliferation, differentiation and the organisation of gene loci within the nucleus. The introduction aims to give an overview of the information required to understand the foundations of studies presented and discussed in this thesis. The transcription or activation of genes occurs in the cell nucleus and requires specific modifications of chromatin. Chapter 1 describes the formation of chromatin and key factors that modify this structure. How transcription is initiated, and influenced by cis-regulatory elements is also discussed. Since the initial characterisation as a transcription factor, many structural and regulatory functions have been attributed to CTCF, as detailed in chapter 2, which imply CTCF is a key regulator of development and cell viability. The haematopoietic system is used in this thesis as a model for investigating the function of CTCF in two distinct lineages. In chapter 3 the development of erythrocytes and T-lymphocytes is introduced. Chapters 4 and 5 describe the experiments used to address questions regarding CTCF function at the b-globin locus and during T-cell differentiation respectively. Published data strongly demonstrate the clustering of cis-regulatory elements into an active chromatin hub (ACH) facilitates transcriptional activation of the b-globin locus. Studies presented in this thesis reveal CTCF is required for normal ACH formation, but suggest additional elements may be required to regulate b-globin gene transcription. By deleting CTCF in T-cells we show that CTCF is necessary for the expansion of T-cells within the thymus. Moreover, CTCF is required for cytokine expression in T-helper type-2 cells and it is shown that CTCF may mediate T-cell receptor signaling. Chapter 6 describes the generation of GFP-CTCF knock-in mice. The data show two distinct dynamic populations of CTCF exist within the nucleus, with the majority of the protein rendered largely immobile. GFP-CTCF is demonstrated to be a functional protein, and can substitute for endogenous CTCF in embryonic stem cells. In the concluding chapter 7, the data presented in this thesis and the future implications of it will be discussed.

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