|
|
The Weatherall Institute of Molecular Medicine |
| Paediatric Molecular Medicine | |
| Department of Paediatrics |
Tel +44 865 222341
email: ann.harris@paediatrics.ox.ac.uk
Post-doctoral research scientists: Dr Joanne Evans, Dr Emma Carter, Dr Sarah Williams, Dr Shih Hsing Leir.
D.Phil. students: Victoria McCarthy, Naila Khodabukus, Pei Ling Wong, Neil Blackledge.
Graduate Research Assistant: Timea Palmai Pallag.
The main interests of the group centre on the basic defect in cystic fibrosis (CF). We are tackling two important questions that are central to the pathology of this disease. First, what are the genetic elements that confer tissue specificity and temporal regulation on expression of the CFTR gene, that when mutated causes CF? Second, why are many organ systems in CF obstructed by mucous secretions? Progress on these topics could make a significant contribution to increasing the efficacy of treatments for CF, either by gene therapy or by pharmacological means.
Funding for our research comes from Cystic Fibrosis Trust, UK, National Institutes of Health USA, Vaincre La Mucoviscidose, France, The Wellcome Trust and the Medical Research Council.
The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. To identify regulatory elements that lie elsewhere in the gene, we mapped DNase1 hypersensitive sites (DHS) in 400kb flanking across the locus. DHS are often, but not exclusively, associated with regulatory regions. We identified 18 DHS that are located 5'and 3' to the CFTR gene and within intronic regions (Smith et al., 1995; Smith et al., 1996; Nuthall., 1999a; Nuthall et al., 1999b; Smith., 2000).
The major focus of current research is to determine which of these DHS contain important regulatory elements for CFTR and how they function together in vivo. Each DHS region has been evaluated for enhancer function using transient reporter gene expression and three of the sites shown to augment CFTR promoter activity (Smith et al., 1996, Phylactides et al., 2002). Cross species homologies between the sheep and human CFTR genes that show a very similar pattern of expression have enabled identification of the conserved sequences (Mouchel et al. 2001). DNase1 footprinting analysis of these sequences, followed by EMSA and supershift experiments have identified several transcription factors that interact with the core elements. (Smith et al., 1996; Nuthall et al., 1999a; unpublished data). Our current studies are aimed at elucidating whether these transcription factors affect CFTR transcription in vivo and if so their mechanism of action.
Further support for the in vivo importance of these DHS regions in regulation of CFTR expression derives from the analysis of a YAC containing the whole CFTR gene. Individual DHS have been removed from the YAC and the expression of CFTR from the modified YAC evaluated in human cells and transgenic mice. In this way the importance of one DHS for normal levels of expression in the colon and small intestine has been established (Rowntree et al., 2001).
We are collaborating with Dr Clare Huxley, Imperial College School of Medicine, London. UK.
One long-term project in the laboratory is to develop an ovine model of CF through gene-targeting of ovine somatic cells followed by cloning. Whilst mouse models of CF have been useful, CF mice do not exhibit the same pathology as humans with the disease. Sheep have very similar anatomy and physiology to humans in organs implicated in CF pathology, such as lung and pancreas. Further, the ovine CFTR cDNA shows a high degree of conservation with human CFTR (Harris, 1997; Tebbutt et al., 1995), exhibits equivalent patterns of temporal and spatial expression and probably shares key regulatory elements for the gene (Mouchel et al., 2000). Through evaluation of the developmental regulation of ovine CFTR gene expression we have identified a window of gestation when CFTR expression is highest in the lung (Broackes-Carter et al., 2002). This may be pertinent to achieving more effective gene therapy for CF.
The failure to clear mucous secretions from epithelial surfaces remains a major problem in CF treatment. Not only are mucin deposits a significant cause of tissue damage but also contribute to the relative inefficiency of gene therapy protocols. We aim to establish the biochemical basis of the CF-associated mucin abnormalities. Twelve mucin genes have been identified all of which share a common structural motif, a tandem repeat (TR) unit at the nucleic acid and amino acid level. The TR contains a high percentage of serine and threonine residues which are the major sites of O-glycosylation. The biochemical properties of a mucin are in large part dependent on the carbohydrate side-chains that are added to the TR by O-glycosylation. We are investigating the O-glycosylation of mucins in the presence of normal or mutant CFTR and the mechanism of release of membrane-tethered mucins (Silverman et al 2001; Parry et al 2001). This is a collaborative project with Professor Anne Dell, Imperial College. London and Dr MA Hollingsworth, UNMC. Omaha, NE. USA.
Since the MUC1 protein probably plays a role in the biological properties of tumor progression, especially the process of metastasis, elucidation of the in vivo mechanisms of regulation of expression of the gene could be of therapeutic value. The promoter of the MUC1 gene has been studied extensively and some cis elements that are important for basal promoter activity have been identified though the available data are largely limited to in vitro assays. We have identified two novel DHS in the MUC1 promoter. These two DHS are detected in vivo in cell lines and in certain tissues from MUC1 transgenic mice that express human transgene. The appearance of one of these DHS correlates with high levels of MUC1 transcription that are characteristic of certain carcinoma cell lines. We are currently investigating the role of these two DHS in MUC1 transcription. This work is in collaboration with Dr MA Hollingsworth UNMC. Omaha, NE. USA and Professor John Hilkens, Netherlands Cancer Institute, Amsterdam.