Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein α in the living pituitary cell nucleus

Richard Day, Ty C. Voss, John F. Enwright, Cynthia F. Booker, Ammasi Periasamy, Fred Schaufele

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein α (C/EBPα) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPα was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPα to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPα was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPα transactivation domain disrupted the redistribution of C/EBPα by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPα, and the truncated C/EBPα still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPα that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPα to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPα are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPα at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

Original languageEnglish (US)
Pages (from-to)333-345
Number of pages13
JournalMolecular Endocrinology
Volume17
Issue number3
DOIs
StatePublished - Mar 1 2003
Externally publishedYes

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CCAAT-Enhancer-Binding Proteins
Cell Nucleus
Proteins
Fluorescence Resonance Energy Transfer
Heterochromatin
Protein Interaction Domains and Motifs
Homeodomain Proteins
Leucine Zippers
DNA
Prolactin
Transcriptional Activation
Microscopy

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein α in the living pituitary cell nucleus. / Day, Richard; Voss, Ty C.; Enwright, John F.; Booker, Cynthia F.; Periasamy, Ammasi; Schaufele, Fred.

In: Molecular Endocrinology, Vol. 17, No. 3, 01.03.2003, p. 333-345.

Research output: Contribution to journalArticle

Day, Richard ; Voss, Ty C. ; Enwright, John F. ; Booker, Cynthia F. ; Periasamy, Ammasi ; Schaufele, Fred. / Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein α in the living pituitary cell nucleus. In: Molecular Endocrinology. 2003 ; Vol. 17, No. 3. pp. 333-345.
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