Dynamic Interactions between Pit-1 and C/EBPα in the Pituitary Cell Nucleus

Abstract

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBP␣) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBP␣ in different ways. The results showed that the first ␣-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBP␣, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.The activation of transcription during development or in response to cell signals involves the transient assembly of multiprotein complexes at specific gene enhancers and promoters. The dynamic assembly of these complexes occurs through networks of protein interactions that are coordinated by sequence-specific DNA-binding factors (8,25). It is through these combinatorial interactions that tissue-specific transcription factors orchestrate eukaryotic gene expression (35). These activities are typified by the homeodomain (HD) transcription factors, a family of structurally conserved proteins that play critical roles in directing cell differentiation pathways during development and in the regulation of programs of gene expression in the differentiated cell types. The central role of these proteins is illustrated by many human diseases, from developmental defects to metabolic disorders that are linked to mutations affecting the HD transcription factors (5). For example, the pituitary-specific HD transcription factor Pit-1 (also called GHF-1 and POU1F1) is a member of the POU family of transcription factors that directs the development of the anterior pituitary lactotrope, somatotrope, and thyrotrope cell lineages (9, 16). Many different point mutations have been identified in the gene encoding Pit-1, and all three Pit-1-dependent cell types fail to develop in patients with inactivating mutations. This results in the syndrome of combined pituitary hormone deficiency (CPHD), a disease characterized by the lack of prolactin (PRL), growth hormone (GH), and thyroid-stimulating hormone beta produced by these cells (6, 31). A clea...

Functional Sequestration of Transcription Factor Activity by Repetitive DNA

“…Pit-1 is a transcription factor that can synergize with C/EBP␣ at pituitary-specific promoters to which both C/EBP␣ and Pit-1 bind (34,35,37,(45)(46)(47). Consistent with the results of our V296A mutant studies, chromatin immunoprecipitation showed that co-expression of Pit-1 reduced the association of wild-type C/EBP␣ with ␣-satellite DNA (Fig.…”

“…4 This precludes the study of the V296A mutant, for instance, in mouse knock-in models. By contrast, some support for a biologic role of C/EBP␣ sequestration was indicated by prior studies showing that the ability of Pit-1 to re-locate C/EBP␣ away from the pericentromeric heterochromatin is disrupted by at least three point mutations in Pit-1 that are found in patients with combined pituitary hormone deficiency (34,35,47). Still, the biologic significance of the intranuclear sequestration of C/EBP␣ transcriptional activity remains to be firmly established.…”

Ligand-Selective Interdomain Conformations of Estrogen Receptor-α

“…On the equipment used in the current studies, 1.18 U of fluorescence from YFP are collected for every 1.00 U collected from an equimolar amount of CFP (determined by expressing both fluorescent proteins on a common translation unit, but spaced far enough apart to permit no energy transfer of CFP to YFP). The loss of donor fluorescence to FRET, which increases nonlinearly with acceptor level, also will affect the acceptor/donor measurement and is more difficult to correct for (32,43). In the current study, the loss of donor to FRET is low, eventually reaching 15% with maximal energy transfer at saturating acceptor levels.…”

Förster resonance energy transfer microscopy and spectroscopy for localizing protein–protein interactions in living cells

“…Choosing a suitable FRET pair is the first step for successful FRET imaging. We are fortunate to have many choices: various fluorescent proteins have been developed and used for FRET imaging to visualize dynamic protein interactions under physiological conditions (26,28,29,(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55). The development of organic dyes with improved photo-stability and excellent spectral characteristics provides additional choices for FRET imaging (27,33,(56)(57)(58)(59)(60), as well as the utility of the emerging field of quantum dots (61,62).…”

“…For example, Dye et al (20) applied a FRET-based flow cytoometric analysis of fusion proteins in live yeast cells to study the dimerization of the wild-type and the mutated Tom70p N-terminal transmembrane domain, and demonstrated that flow cytoometry combined with FRET is a powerful tool for studying proteinprotein interactions in a large number of individual cells. FRET microscopy imaging can provide spatial and temporal information of protein interactions in living cells under physiological conditions to address fundamental biological questions (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Confocal FRET microscopy was used to track the internalization of transferrin receptor-ligand complexes in live cells (27,33).…”