Unit: Genetics of Differentiation
Director: Mary C. Weiss
The unit studies the mechanims implicated in the regulation of expression of hepatic genes and in the roles of the transcription factors that are necessary for the establishment and maintenance of hepatic differentiation. In addition, we have established new experimental models of liver cell differentiation, and are attempting to identify transcripts that are specifically induced when hapatocytes are infected by sporozoites of Plasmodium berghei.
The Unit of Genetics of Differentiation uses cell genetics to analyse the roles of different Liver Enriched Transcription Factors (LETF) in the establishment and maintenance of the differentiated hepatic phenotype. It has been shown that in dedifferentiated rat hepatoma cells, the forced expression of HNF4α cDNA is sufficient to provoke the re-expression of genes that are considered markers of hepatic differentiation. In addition, in some cell lines of hepatic origin which fail to express either the LETF or hepatocyte differentiation, the forced expression of either HNF1α or of HNF4α cDNA is sufficient to ensure the expression of the endogenous genes for both factors, indicating the existence of a reciprocal regulatory loop between these two key transcription factors.
At present, our efforts are concentrated on the study of HNF4α, a transcription factor of the nuclear receptor family which has been identified as a key factor for execution of the liver differentiation program. The HNF4α gene has two alternative promoters whose use is regulated during the course of liver development. The protein resulting from use during embryogenesis of the distal promoter, HNF4α7, shows avid transactivation in tranfection tests of genes that are expressed early during development. In contrast, HNF4α1 is produced in abundant fashion only at birth, and this protein shows strong activity on promoters of genes that are expressed neonatally. Thus, the timing of expression and the activity of the two HNF4α isoforms are complementary.
To analyse the roles of the two isoforms of HNF4, α1 and α7, we have used directed mutagenesis in the mouse to create animals that can express only one of the two isoforms, but under control of both of the respective promoters.
To understand the basis for the differences in transcriptional activity of the HNF4α1 and HNF4α7 isoforms, which differ only in their N-terminal amino acids, the two transactivation domains known for nuclear receptors were analysed using tests of in vitro interaction as well as co-transfection assays. It was found that the region corresponding to the AF-1 of HNF4α7 is devoid of activity while that of HNF4α1 showed the anticipated activity, that was increased by interaction with GRIP and CBP. For both isoforms the AF-2 domain was able to mediate interactions with GRIP-1, p300 and the corepressor SMRT. However, the repression imposed by SMRT was less robust for HNF4α7 than for HNF4α1. Finally, both isoforms, in association with SMRT, are able to recuite HDAC1 and 4, and when the three molecules are associated, a larger quantity of HNF4α1, but not of HNF4α7, is fixed. Moreover, in a test of transfection in the presence of an inhibitor of HDACs, a dramatic increase of reporter gene activity is observed for HNF4α1 but not HNF4α7. These results imply that HNF4α1, the isoform found in adult liver, is more subject to regulation by interaction with coactivators and corepressors than the HNF4α7 isoform that is present mainly in embryonic and fetal liver.
We have continued to study bipotential cell lines obtained from embyronic liver of the mouse. While the first lines to be studied here were obtained from transgenic mice expressing an activiated form of human c-Met in the liver, our recent attempts to obtain them from non-transgenic mice have been successful. Thus, we now have bipotential liver lines from a large variety of mouse lines. In addition, we have improved the methods for obtaining their differentiation into cholangiocytes and hepatocytes. Hepatocyte functions such as apolipoproteins, albumin, AFP, alcohol dehydrogenase and aldolase B are induced upon culture for a few days in the form of aggregates. When cultured in Matrigel, not only are cholangiocyte/oval cell markers such as CD34, c-kit, integrin b4, connexins and GGTIV induced, but morphogenesis into bile duct units is observed. At present, we are using these techniques to analyse cell lines obtained from mouse embryos carrying targeted mutations in transcription factors important for liver dvelopment. These studies will permit us to clarify the roles of these factors in the maintenance of the bipotential phenotype and in the execution of the cholangoicyte and hepatoctyte programs. Finally, these cell lines are being tested for their capacity to participate in liver regeneration, by differentiating into mature hepatocytes and cholangiocytes.
Keywords: Hepatic differentiation, liver-enriched transcription factor, co-regalutor, bipotential cells