Measuring dynamic network interactions that control pituitary-gene expression

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through interactions with other nuclear proteins, control patterns of gene expression during development. For example, the combinatorial interactions of the HD protein Pit-1 with transcription factors such as the CCAAT/enhancer binding protein alpha (C/EBPalpha), and with coregulatory proteins (coactivators and corepressors), control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. These same pituitary cell-types fail to develop in human patients with mutations affecting the Pit-1 gene, resulting in combined pituitary hormone deficiency (CPHD). Our previous studies showed that Pit-1 redistributed C/EBPalpha from regions of heterochromatin to the euchromatic subcompartment, and this activity was correlated with the cooperative activation of the prolactin gene. In this proposal, I describe biochemical, live-cell imaging and in vivo kinetics studies that provide new insight into the dynamic network interactions of Pit-1 with C/EBPalpha and coregulatory proteins in the pituitary-cell nucleus. Like most chromatin-associated proteins, Pit-1 and C/EBPalpha are dynamic within the nuclear compartment, and their mobilities reflect their transient interactions with chromatin and with a variety of different protein partners. In this regard, coexpression of Pit-1 caused a significant reduction in the intranuclear mobility of C/EBPalpha. Moreover, two different CPHD-linked point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions with C/EBPalpha or the coregulator complexes in different ways. My findings support a model where proteins move independently within the nuclear compartment, and stochastically associate at specific intranuclear sites as part of metastable complexes. The probability that these proteins assemble in a particular region of the cell nucleus was related to the dominant chromatin-binding activities of the different protein partners, supporting the view that the repositioning of transcription factors and coregulators between nuclear subcompartments represents an important mechanism for directing changes in cell-specific gene expression. My study also suggests an important link between the mislocalization of transcriptional complexes and disease processes. Although my research focused on pituitary-specific transcriptional networks, these findings are applicable to the molecular mechanisms that control the intranuclear organization of different classes of transcription factors.