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/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. 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/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α 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 HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.
The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with
exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy
transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring
dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps
changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic
protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined
with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving
transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The
heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin
through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α
as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor
CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in
mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic
interactions in pituitary specific gene regulation.
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