Using Isothermal Titration Calorimetry for Biophysical Characterization of Chromatin-Binding Proteins

Biophysical characterization of epigenetic protein interactions with chromatin using Isothermal Titration Calorimetry

Abstract

Epigenetic regulation of genomic DNA for gene expression is important in cellular differentiation and the development of an organism. Epigenetics also contributes to human diseases. This white paper summarizes how Isothermal Titration Calorimetry (ITC) is used for characterization of proteins involved in epigenetic regulation.

Introduction to epigenetics

Epigenetics is the study of heritable changes in gene expression caused by nongenetic mechanisms, without alterations in gene structure or DNA sequence. The epigenetic state of a cell evolves during the cellular differentiation and development of an organism, and epigenetic changes are linked to cellular reprogramming. Because epigenetic mechanisms may also be responsible for the integration of environmental responses at the cellular level, they potentially play an important role in the development of some diseases.

Epigenetic regulation of gene activity is complex and not yet fully understood, and involves transcription factors, growth factors and perhaps hormones. Epigenetic processes also involve the modification of chromatin (Figure 1). A histone octamer, composed of two copies of each of the histone proteins H2A, H2B, H3, and H4, is wrapped by a strand of 145-147 bp DNA, forming a nucleosome core. Multiple nucleosomes pack together to form chromatin.

Epigenetic events likely involve covalent modifications of histones, DNA and RNA, as well as chromatin remodeling, micro-RNA mediation, and other changes to chromatin structure. The flexible N-terminal tails of histones contain a range of site-specific post-translational modifications (PTMs) called "marks," including methylation and acetylation of lysine, methylation of arginine, and phosphorylation of serine, threonine and tyrosine residues. The PTMs on histone tails form specific patterns, and are added, read and removed by specific enzymes in a sequence- and modification-specific manner, resulting in additional marks.The existence and/or absence of marks on histone tails provides unique docking sites for specific binding and/or release of downstream effector proteins, resulting in diverse biological functions including transcription regulation, cell cycle control, differentiation and apoptosis.

In addition to histone tail modifications, DNA methylation at the 5 position of the cytosine base constitutes another common covalent epigenetic mark, which can contribute to gene silencing.