| Summary: | Superenhancers are clusters of putative enhancers, densely occupied by histone 3 lysine 27 acetylation, transcription factors, and coactivators such as the mediator complex. There is a tendency for superenhancers to regulate key cell-type-specific genes, and their dysregulation is often associated with disease. It remains unclear whether superenhancer constituent elements work independently or collaboratively and whether they have distinct roles in activating expression of their cognate genes. During my DPhil, I address these two questions by studying a model mammalian superenhancer at the mouse α-globin locus, active exclusively in erythroid cells. I sought to determine how the five constituents (R1, R2, R3, Rm, R4) of the mouse αglobin superenhancer exert control over α-globin gene expression during erythropoiesis. Firstly, I tested the sufficiency of the previously described strongest αglobin superenhancer constituent (R2) to independently activate α-globin expression; this entailed characterising, in detail, a mouse model in which the other four α-globin superenhancer elements have been removed from the native locus. Surprisingly, I show that this strong enhancer is incapable of driving the expected level of expression, independently. Secondly, following synthetic biology and genome editing techniques in mouse Embryonic Stem Cells (mESCs), I rebuilt the native α-globin superenhancer in all informative combinations, starting from an enhancer-less baseline in which all five constituents have been removed. Examination of molecular phenotypes of erythroid cells derived from the engineered mESCs revealed a complex relationship between inequivalent constituents which cooperate in additive, synergistic, and redundant fashions. I also uncovered a novel class of regulatory element within the αglobin superenhancer, which I named facilitators. Unlike canonical enhancers (R1, R2), facilitators (R3, Rm, R4) have no intrinsic enhancer activity. However, they are necessary to potentiate canonical enhancers, in order to attain optimal levels of target gene expression. Lastly, by comparing the impact of the three facilitators on gene expression, I discovered a functional hierarchy that seems to be position-dependent. Ultimately, I have rigorously dissected the α-globin superenhancer and described a new class of regulatory element, which I named facilitators, that were previously mistaken for weak enhancers. Furthermore, I unravelled a necessary mode of cooperation manifested between two types of superenhancer constituent elements, canonical enhancers and facilitators, highlighting an emergent property of a superenhancer.
|
|---|