Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

Amanda P. Siegel, Nicole M. Hays, Richard N. Day

Research output: Contribution to journalArticle

10 Scopus citations


The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPa), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1a. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPa is sufficient for the interaction with HP1a in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1a and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1a, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1a W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1a or HP1aW174A. The functional significance of these interactions is discussed.

Original languageEnglish (US)
Article number025002
JournalJournal of Biomedical Optics
Issue number2
StatePublished - Jul 15 2013



  • fluorescence correlation spectroscopy
  • fluorescence lifetime imaging microscopy
  • fluorescent proteins
  • Förster resonance energy transfer microscopy
  • protein interactions

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

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