Peripheral Localization and Regulation of the Murine β-Globin Locus.

The interphase cell nucleus is structurally and functionally compartmentalized, making the subnuclear position of genes an important determinant of their activity. During cellular differentiation, as tissue-specific expression programs unfold, dynamic reorganization of the nucleus positions sets of genes in active or repressive compartments. The nuclear periphery has emerged as an unusually complex compartment in this process. While it is marked by facultative heterochromatin and has been considered primarily as a repressive compartment, recent work suggests that active genes may also associate with the periphery. Moreover, the nuclear envelope is riddled with nuclear pore complexes, the gateways for molecular exchange between the cytoplasm and the nucleus, resulting in substantial traffic through this compartment. Using murine erythropoiesis as a model system, our recent three dimensional analysis of the positioning of the β-globin locus revealed that, when inactive in undifferentiated embryonic stem cells and erythroid progenitor cells, the locus is positioned predominantly at the nuclear periphery and appears to contact the lamina. This association is lost with progressing erythroid maturation, and the locus is repositioned towards the nuclear interior concomitant with increasing β-globin gene expression. Importantly, however, β-major globin expression begins at the nuclear periphery prior to relocalization, suggesting that associations of the locus at the periphery may shift from repressive to activating complexes. We are investigating the interactions of the β-globin locus with the nuclear periphery by two approaches:

1. Using enhanced imaging permitted by Cryo-ImmunoFISH, we are analyzing the position of the locus relative to specific components of the nuclear lamina and nuclear pore complexes at distinct differentiation stages.

2. In addition, we are probing the physical interactions of the locus with the periphery by biochemical means.

To this end we are using ChIP-chip to identify lamina associated proteins binding the β-globin locus and to determine what sequence elements within the locus mediate these interactions. Ultimately these experiments will shed further light on mechanisms regulating β-globin gene expression during erythropoiesis and how stage-specific nuclear localization contributes to this process.