Nucleosomal DNA is highly distorted and partially occluded
from the solvent due to its tight interaction with the histone
octamer. Thus, nucleosome architecture greatly affects the
accessibility of nucleosomal DNA for global and specific
regulators. We are investigating recognition of nucleosomes by
small molecules, viral proteins, and transcriptional regulators,
using fluorescence anisotropy, fluorescence resonance energy
transfer, analytical ultracentrifugation, gel electrophoresis,
and x-ray crystallography. In particular, we are interested to
what degree nucleosome structure adjusts to better accommodate
interacting factors.
In collaboration with the
Dervan and
Gottesfeld laboratories,
we have demonstrated that nucleosomal DNA is quite accessible
for molecular recognition. Pyrrole-imidazole polyamides bind
nucleosomal DNA with high affinity and specificity, and evoke
distinct structural changes in nucleosomal DNA. These results
demonstrate that nucleosomal DNA is capable of adjusting (within
limits) to provide an optimal binding environment for a ligand
while still remaining tightly bound to the histone octamer. We
have further shown that polyamides that bind nucleosomal DNA
severely limit the ability of the histone octamer to reposition
with respect to the DNA, thereby completely inhibiting in vitro
transcription.
We have recently expanded our studies to protein transcription
factors. Using a combination of methods, most notably x-ray
crystallography, and fluorescence resonance energy transfer
between defined regions within the nucleosome, we investigate
the structural changes that are inflicted upon the nucleosome
and the transcription factor as a consequence of binding to
nucleosomal DNA. Effects on higher order structure are also
studied using analytical ultracentrifugation and atomic force
microscopy.
We are also investigating how the nucleosomal surface may serve
as a binding platform for cellular and viral factors. We have
started by studying how Kaposi’s sarcoma herpes virus protein
LANA enables the viral genome to tether onto chromosomes so that
virus is not lost from cells. The mechanism by which LANA
latches onto chromosomes was previously unknown. We have found
that LANA engages histones H2A and H2B to dock onto chromosomes
by binding to the nucleosomal surface via a tight hairpin motif.
This study (which is the result of an on-going collaboration
with
Dr. Kenneth Kaye) unequivocally demonstrates how a highly
structured nucleosomal surface acts as an interaction platform
for molecular recognition.
