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

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/EBP alpha), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1 alpha. 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/EBP alpha is sufficient for the interaction with HP1 alpha in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1 alpha 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 Frster resonance energy transfer (FRET). A point mutation in HP1 alpha, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1 alpha W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1 alpha or HP1 alpha W174A. The functional significance of these interactions is discussed. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.18.2.025002]