The genetic material of all higher organisms from yeast to mammals is organised
in the cell nucleus as a nucleoprotein complex called chromatin. The fundamental
repeating unit of chromatin, which covers nearly the entire DNA, is the nucleosome.
Each one comprises eight highly conserved protein subunits that sequester
approximately 146bp of DNA. Nucleosomes facilitate the highly condensed packaging
of DNA, most obvious in metaphase chromosomes, and also permit non-histone protein
factors access to the DNA in order to facilitate DNA replication, transcription and repair.
For temporally and spatially specific gene activation to occur, chromatin
remodeling factors, transcription factors and RNA polymerase and its associated factors
must act in concert with the underlying nucleosome environment to effect transcription.
In some instances, this has shown to be a complex relationship. Nucleosomes are stably
positioned over transcription factor binding sites in some genes. This can prevent access
and therefore repress gene activation. In other genes, a positioned nucleosome is
required to wrap up DNA between separate transcription factor binding sites. Bringing
the sites together allows the binding factors to act cooperatively in initiating
transcription. Therefore, nucleosomes that are positioned over a specific DNA sequence
can have an instrumental role in gene regulation.
To date, there have only been limited studies on the nucleosomal organisation of
genes in their natural environment. The majority of these studies have concentrated on
short regions of positioned nucleosomes spanning either repetitive DNA or the promoter
regions of specific genes. However, nucleosome positioning over an entire gene domain
may have a significant impact on its regulation and compaction. I have mapped the
nucleosomal organisation over lOkb of a tissue specific, temporally regulated gene using
the enzymatic probe, micrococcal nuclease and the chemical probe, cuprous
phenanthroline. The ovine p-lactoglobulin (BLG) gene studied has a well characterised
developmental profile, a minimal transcriptional domain and has been used extensively
as an expression cassette in transgenic animals to drive heterologous gene transcription
When the gene is inactive, in the liver, it displays a tightly defined array of
positioned nucleosomes that modulate between two specific phases over the gene
domain. A similar, less tightly defined array is present when the gene is active, in the
mammary gland, except over the promoter and actively transcribing regions. The same
arrays arc present over the BLG promoter region in transgenic mice in both active and
inactive states. A monomer extension reaction provides in vitro evidence of the
positioning signals that are determined by DNA sequence alone. These show an
interesting correlation with the in vivo results.
A number of other milk protein genes have a similar pattern of key transcription
factor binding sites over their promoter regions. If the nucleosome positions were
conserved in these genes, with respect to these binding sites, it might suggest a role for
positioned nucleosomes in their regulation. A total of three genes, each in two different
organisms, have been analysed to test for a correlation.