Transcriptional Control During Hematopoietic Development

A cell’s identity is primarily determined by the proteins it produces and therefore by the genes it expresses. During development, correct cell fate specification and determination therefore requires a strictly controlled upregulation or downregulation of lineage-specific gene expression. The experimental work described in this thesis aimed to contribute to unraveling the complex process of such gene expression regulation. The first part of Chapter 1 gives a general introduction on the mechanism of gene expression regulation, with a main focus on the first step in this process, i.e. gene transcription. This first part is followed by a general introduction on hematopoietic development and differentiation, which is a widely used model system for studying gene expression regulation during cellular differentiation. This part briefly discusses the origin of hematopoietic stem cells (HSCs) and their terminal differentiation towards the different blood cell lineages, with a specific focus on erythropoiesis. HSCs are known to originate in the Aorta-Gonad-Mesonephros region of the embryo, where they derive from specialized intra-aortic hematopoietic clusters (IAHCs). However, at present, the precise mechanism of HSC specification in these clusters of cells is not known. Chapter 2 describes the results of our study on the role of the IAHC cells in the development of HSCs. Chromatin structural conformation plays a central role in gene expression regulation, with multiple regulatory elements being located at far distance from their target genes, and often regulating gene expression via the formation of chromatin loops. Chapter 3 of this thesis discusses the role of distal regulatory elements and chromatin conformation in regulating gene transcription. In addition, it discusses the role of genomic alterations in our noncoding ‘regulatory’ genome in disease susceptibility and etiology, and discusses several novel therapeutic strategies to target such molecular diseases. Chapter 4 describes the optimization of the chromatin conformation capture (3C) technology for use in semi-high throughput multiplexed next-generation sequencing analyses. Multiplexed 3C-seq, provides a tool for analyzing the fine-scale chromatin structural conformation and can be used to study the role of chromatin structure on gene-specific expression regulation. Chapter 5 describes the results of our study on the transcriptional control of murine Bcl11a, with a specific focus on the role of the erythroid-specific LDB1 transcription factor complex in this regulation. Data from this study may contribute to unraveling the different mechanisms of transcriptional control used to ensure correct and accurate transcription levels and patterns during hematopoiesis. Moreover, as BCL11a itself is known to be a repressor of γ-globin expression, Bcl11a repression has become an interesting therapeutic strategy for β-hemoglobinopathies in which elevated γ-globin levels have an ameliorating effect on disease phenotypes. Data from this study may therefore provide new avenues for therapeutic treatment of these globin-related diseases. Finally, Chapter 6 presents a general discussion on the experimental data presented in this thesis. It highlights important findings and aims to place this data in a broader perspective by discussing its contribution to the general understanding of the mechanism of gene transcriptional control. It discusses how these results can contribute to the ultimate goal of unraveling the complete mechanisms of hematopoietic development and how these may contribute to the development of new treatments for hematological diseases.