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

Amanda P Siegel,Richard N Day,Nicole M Hays

doi:10.1117/1.jbo.18.2.025002

Amanda P Siegel, Richard N Day + Show 1 more

Open Access

https://doi.org/10.1117/1.jbo.18.2.025002

Copy DOI

Abstract

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.

Highlights

There is a clear link between epigenetic dysregulation and the progression of many diseases including cancer, cardiovascular disease, diabetes, and obesity, but the mechanisms that control epigenetic signaling is poorly understood
Our results argue that the functional interaction of HP1α with methylated chromatin is necessary for a strong interaction with basic region and leucine zipper (BZip) domain of C/EBPα, and this interaction could reflect a commonality in the activities of these proteins
We used the combination of fluorescence correlation spectroscopy (FCS), fluorescence correlation and cross-correlation (FCCS), and Förster resonance energy transfer (FRET)-FLIM to investigate the dynamic interactions between C/EBPα and HP1α in the nuclei of living pituitary cells

Summary

Introduction

There is a clear link between epigenetic dysregulation and the progression of many diseases including cancer, cardiovascular disease, diabetes, and obesity, but the mechanisms that control epigenetic signaling is poorly understood. It is well established that the epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by heterochromatin protein 1 (HP1).[1] While HP1 is principally known for directing the assembly and maintenance of heterochromatin through direct interactions with the histone methyltransferases (HMTs), it has become increasingly clear that HP1 has much broader roles in the regulation of gene expression.[1,2,3,4] Recent studies suggest the HP1 proteins are positive regulators of gene transcription,[3] and play roles in DNA replication[5] and repair.[6] A major issue in the field is to determine how the HP1 network interacts with sequence specific transcription factors, and how these interactions function to target chromatin remodeling activities to specific gene promoters

Methods

Results

Discussion

Conclusion