1.  INTRODUCTION

     The accurate switching ‘on’ and ‘off’ of gene expression is central for life with each gene having its own specific spatial and temporal expression pattern. To ensure this pattern is faithfully adhered to several layers of regulation are imposed within the cell. Over the past two decades there have been great advances in our understanding of how soluble transcription factors regulate gene expression. With this knowledge has come an increased awareness of the importance of the whole nuclear environment within which genes are regulated. Gene expression takes place in the context of chromatin, a condition that many think contributes to the developmental and spatial control of transcription. Clearly a full understanding of how a gene is regulated must incorporate knowledge of the chromosomal environment in which it is contained.
       We have focussed our effort on investigating mammary gene expression. The mammary gland represents a differentiated tissue which expresses a specific set of genes in response to a variety of hormonal, cellular and extracellular matrix derived signals. It is also the only organ in which the majority of development takes place in the adult and which can undergo successive cycles of development and regression. In addition, the mammary gland is an important organ from an agricultural perspective (milk production) and can be harnessed for new genetic biotechnologies.

2.  β-LACTOGLOBULINGENE

    In most mammals, the major whey protein is β-lactoglobulin (βlg). The function of βlg is unconfirmed although it shares considerable similarity to retinol-binding protein and other lipocalins and therefore may transport small hydrophobic molecules. In ruminants, this protein represents a marker for tissue-specific, temporally regulated gene expression in the mammary gland. Numerous studies in a variety of cell-based systems have shown that βlg expression is regulated by a complex interaction of hormones and growth factors in association with cell-cell and cell-extracellular matrix interactions. The major lactogenic stimulus prolactin, activates βlg expression through a cytoplasmic signalling cascade ending in the activation of the transcription factor STAT5. There are three binding sites for STAT5 within the ovine blg gene promoter (Fig. 1).Activation of βlg gene expression is accompanied by changes in the chromatin structure surrounding the promoter region. No DNaseI hypersensitive sites are present in the liver where the gene is not expressed, while distinct sites are present in the lactating sheep mammary gland. Additional mammary specific hypersensitive sites are detected within the first two introns prior to over βlg transcription. Analysis, both in cell culture and transgenic mice, indicates that only the hypersensitive site located at the promoter, termed HSIII, is essential for expression in mammary cells.

Nucleosome organisation of the β-lactoglobulin gene
              


Fig. 1 .The ovine βlg gene contains 7 exons (black boxes) within about 5 kb of genomic sequence. The entire sequence is known (X12817). Mammary specific DNaseI hypersensitive sites are depicted as arrows with roman numerals . Specific transcription binding sites are shown in the enlarged promoter region; open circles, STAT5; filled circle, TATA-box.DNaseI hypersensitivity at HSIII parallels this expression profile, with minimal cutting before mid-pregnancy and substantial cutting thereafter and during lactation. This temporal pattern of DNaseI hypersensitivity parallels the differential activation of complexes containing the prolactin induced transcription factors STAT5a and STAT5b. Specifically, there is a marked increase in STAT5a relative to STAT5b during late pregnancy which suggests a major role for STAT5a in regulating βlg expression. In support of this, STAT5a null-mice indicate that this transcription factor is the principal mediator of the lactogenic signal. Nevertheless, STAT5a interaction with the βlg promoter is not essential for tissue-specific expression of βlg12 Therefore, other mechanisms must be involved in establishing a mammary-restricted transcription factor complex upon the βlg promoter.

3.  NUCLEOSOME MAPPING OF β-LACTOGLO- BULIN PROMOTER

     The positioning of nucleosomes contributes to both the structure and function of chromatin and has a decisive role in controlling gene expression. A major determinant of nucleosome positioning, and therefore distribution, is the DNA sequence itself. Other components may modulate DNA-directed nucleosome positioning, but must act in conjunction with, rather than independently of the DNA sequences. In most studies of sequence-directed nucleosome positioning, only short regions of DNA have been analysed in any detail. Understandably, the tendency has been to focus in the immediate vicinity of regulatory DNA sequences. Only one study has addressed an entire gene.We have mapped the in vivo nucleosomal profile for about 10 kb of contiguous genomic sequence encompassing the ovine blg gene. Two probes of nucleosomal organisation have been used. The first relies on the ability of the enzyme MNase to preferentially digest DNA in the linker region between two nucleosomes. The second approach involves cuprous phenanthroline probed mercaptopropionic acid cleaved chromatin. Again cleavage is predominately in the linker region between nucleosomes. Both methods clearly indicate that a periodic nucleosome pattern based on 180 bp is present on both the inactive and active blg gene. Furthermore, reflecting gene expression state, there are distinct differences between the active and inactive chromatin. In addition, a specific relationship between nucleosome positioning over the promoter region and the location of known transcription factor binding sites is evident (Fig. 3).To further define the nucleosomal profile of blg we have mapped in vitro reconstituted,   sequence directed nucleosomal positioning.  This has been performed using the monomer extension assay developed by Dr, Jim Allan (University of Edinburgh) and optimised for βlg by Simon Boa. The in vitro map essentially supports the in vivo map and offers the opportunity to identify sequence determinates of nucleosomal positioning.


                                            

Fig. 3. Alternative nucleosome positions relative to STAT5 binding site (open circle). Thus, the STAT5 site resides either within the linker region between two adjacent nucleosomes or near the nucleosome dyad.

4.  PROPOSED MODEL FOR TRANSCRIPTION COMPLEX FORMATIONAT β-LACTOGLO-BULIN PROMOTER
    
      From the DNaseI and nucleosomal mapping data we propose a co­operative model for transcription complex formation at the ovine β1g promoter. This model revolves around the precise positioning of a nucleosome within the promoter (Boa et al., manuscript in preparation). Furthermore, STAT5 probably fulfils a late function to stabilise the transcription factor complex (Whitelaw et al., manuscript in preparation), thus ensuring efficient transcription.