The Trouble with Triples: Elucidating the Behavior of Trinucleotide Repeats in Chromatin

Abstract

Trinucleotide repeats (TNRs) occur throughout the genome and their expansion can affect varied cellular processes such as gene expression, mRNA processing, and protein folding. TNR expansion, facilitated by formation of stable non-canonical secondary structures (e.g. hairpins), has been linked to several neurodegenerative diseases, such as Huntington's Disease, Myotonic Dystrophy (both caused by expansion of CAG•CTG repeats), and Fragile X Mental Retardation (caused by expansion of CGG•CCG repeats). TNR structure and expansion has been studied in vitro using both oligonucleotides and TNRs incorporated into plasmids; however, genomic DNA is packaged into chromatin, and little is know about the effect of packaging on the ability of repetitive DNA to expand. Previous studies have shown that disease-length, expanded CAG•CTG repeats readily incorporate into the basic unit of chromatin packing, the nucleosome core particle (NCP), composed of 146 base pairs of DNA wrapped around a core of eight histone proteins. However, long CGG•CCG repeats exclude NCP formation. Here, we seek to investigate not only the properties associated with shorter repeats (those that have not expanded) incorporated into NCPs, but also the effect of the flanking, disease-associated gene sequence. To assess the global forces regulating interaction between the TNRs and the histone core, we performed competitive nucleosome exchanges to determine the efficiency with which various TNR sequences incorporate into NCPs. We then used enzymatic and chemical probing to determine the effect of histone occupancy on the TNRs, including the formation of possible non-canonical structures. Understanding the intricacies underlying the interaction between unexpanded TNRs and the histone core is important to understanding how TNRs expand in the genome.