Our detailed objectives were to study:
(1) structure and function of HNF1 and 4 and the domains that control DNA binding and their ability to dimerize and to activate transcription alone or with cofactors.
(2) function of HNF1, vHNF1 and HNF4 in mice by means of classic `knock-out' experiments.
(3) regulation of HNF1 and 4 in somatic cell hybrids.
(4) regulation of the promoter affinity of HNF1 and 4 for liver-expressed genes, including the factor IX gene.
There has been good progress on all 4 objectives of our proposals, although the most high-profile work has been the generation of the HNF1 knock-out mouse and its extensive characterization. This is leading to further collaborative work, which could not have been envisaged at the start.
HNF1 and vHNF1 knock-out mouse
HNF1 knock-out mice are viable but fail to thrive, suffering from a progressive wasting syndrome and death shortly after weaning. The transcription of target genes in the liver, e.g. albumin, alpha1 antitrypsin, etc is reduced, while the gene coding for phenylalanine hydroxylase (PAH) is totally silent, giving rise to phenylketonuria (PKU). The mice also suffer from severe renal proximal tubular dysfunction reminiscent of Falconi syndrome in Man. The consequent loss of glucose, amino-acids and water is the probable cause of death. It is hoped that vHNF1 knock out mice, which are under construction, will be available shortly for detailed characterization
2. Regulation of PAH
The regulation of the PAH gene is hormone inducible and somewhat unusual in that there is an upstream enhancer (-3.5 kb) completely dependent on HNF1 expression. Maximum inducibility requires the integrity of HNF1 & C/EBP sites.
Regulation of HNF1 & 4 in somatic cell hybrids
An expression HNF4 cDNA construct, when introduced stably into a dedifferentiated H5 hepatoma cell line, partially restored the hepatic phenotype e.g. expressing endogenous HNF1, transthyretin, etc confirming the importance of HNF4 in hepatocyte differentiation. However, other experiments in somatic cell hybrids suggest that there are additional regulatory factors besides HNF1 and 4. High frequency apoptosis occurs in some hepatoma hybrids providing a new model to assess factors controlling cell death.
4. Regulation of the factor IX & VIII genes by HNF1 &4
Taking advantage of naturally occurring point mutations in the factor IX promoter/enhancer, a new HNF4 binding element has been characterized centred at nucleotide -6, which explains the reason for the haemophilia B in such patients. In addition, ARP1 may repress the factor IX gene at this site and others. The factor VIII proximal promoter/enhancer region has been newly characterized. A proximal promoter/enhancer of 200 nucleotides contains the minimal essential regulatory region. HNF1 and 4 sites are present, as well as elements specific for other ubiquitous and liver-expressed genes (C/EBP).
MAJOR SCIENTIFIC BREAKTHROUGHS:
Our studies on the HNF1 knock-out mice are a significant 'breakthrough'.
Funding SchemeCSC - Cost-sharing contracts