Chromatin looping and epigenetic regulation at the maize b1 locus

In this thesis, the effect of epigenetic regulation on long-range chromatin looping is studied. As a model system we used two maize b1 epialleles involved in paramutation. Paramutation entails a trans-interaction between two alleles whereby one allele heritably changes the expression level of the other. At the b1 locus this occurs between B-I and B’; B-I expresses the b1 gene at a high level, B’ at a low level. Chapter 1 presents a general overview of paramutation across kingdoms and particular attention is paid to the common features of various paramutation phenomena. Chapter 2 describes the optimalization of the Chromosome Conformation Capture (3C) technique for plant genomes. 3C is an excellent technique to uncover physical, long-range chromatin interactions. 3C had previously not been implemented on plant tissue and protocols established for mammalian cells could not be applied directly to plant material, but required plant-specific adjustments. The optimalization process was technically demanding and a crucial part of my PhD studies. In Chapter 3 the 3C technique is applied to the b1 locus. Previous reported results suggested the existence of long-range interactions at the b1 locus. Here, we identified tissue-specific as well as expression level-specific long-range interactions. Interestingly, the chromosome configuration differs between B-I and B’. The results indicate that several sequence regions are involved in mediating high expression. Further analysis suggested the existence of regulatory sequences involved in high expression. Both B-I and B’ are epigenetically regulated and to unravel the underlying regulatory mechanisms, mediator of paramutation (mop) mutants affecting paramutation and the B’ expression level have been used. Chapter 4 describes the study of various aspects involved in the epigenetic regulation of B’ in a mop1-1 mutant background. In Chapter 5, the spatial organization of the b1 locus in two additional mop mutants is studied. The results on mop2-1/Mop2 and mop3-1 indicate that defined chromosome conformations are associated with a high and low b1 expression status. Chapter 6 is the general discussion. The information on the epigenetic regulation of B-I and B’ described in the previous chapters is put into perspective. Furthermore, we draw models for paramutation, the process in which B-I and B’ are involved.