Isolation and characterization of satellite heterochromatin

Sonia Jinane Nasr, Purdue University

Abstract

In the eukaryotic nucleus, long DNA molecules have to be packaged in a limited space, where they are maintained in a well-organized structure called chromatin. Chromatin displays a dynamic conformation involved in the modulation of important genetic activities, such as transcription, replication, recombination, and repair. Both the DNA structure and the protein composition of chromatin play important roles in the folding and unfolding of chromatin. The study of these structural transitions would be greatly aided by the isolation of a homogeneous chromatin sample, with well-defined characteristics. This study describes the purification of polynucleosomes containing a satellite sequence from the meal worm beetle T. molitor. Satellite DNAs are highly repeated sequences located in regions of transcriptionally inactive heterochromatin. The T. molitor satellite, representing 50% of the genome, was isolated and sequenced. The molecular characterization of this DNA, by computational and electrophoretic analyses, showed that it exhibits sequence-dependent curvature, in two bending centers. This structure has recently been shown in our lab to direct nucleosome positioning, a mechanism implicated in transcriptional repression. The chromatin containing the satellite DNA was selectively isolated through restriction digestion by EcoRI, known to cut the satellite DNA into 142 bp monomer units. However, upon digestion of the chromatin with EcoRI, the smallest nucleoprotein fragment generated contains a tetramer of the satellite (568 bp), not a monomer. Subsequently, the nucleosome repeat lengths of satellite heterochromatin and of the total chromatin were determined. The linker DNA, of 35 bp average length, was incorporated as a key component in a model that would explain the peculiar digestion pattern of the chromatin by EcoRI. In this model, the EcoRI would only cut the chromatin when its recognition sequence is in the linker DNA. Accordingly, nucleosome mobility was induced and the effect of a protein, linker histone H1, on this process was investigated. We concluded that histone H1 immobilizes the core nucleosomes on the DNA, in addition to its known function in maintaining the higher-order structure of chromatin in the 30 nm fiber. Subsequently, the satellite heterochromatin was fractionated on nondenaturing polyacrylamide gel electrophoresis and on sucrose density gradients. This led to the purification of satellite oligonucleosomes of a well-defined length and DNA sequence and structure. These polynucleosomes were found to contain the full complement of core histones and linker histone H1. In addition, they were found in their native structure, as assessed by nuclease digestions. The results of this study represent the first direct characterization of isolated native oligonucleosomes containing a specific bent sequence.

Degree

Ph.D.

Advisors

Anderson, Purdue University.

Subject Area

Molecular biology

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