Nucleosomal organisation over the ovine β-lactoglobulin gene

Abstract

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.