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Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition.

ABSTRACT: Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation. Many of these zinc-finger domains are involved in DNA binding. This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition. (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition. The protein-DNA interaction was examined in greater detail by using a methylation-interference assay. The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide. This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers.

SUBMITTER: Green A

PROVIDER: S-EPMC1219559 | biostudies-other | 1998 Jul

REPOSITORIES: biostudies-other

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Project description:Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1?). However, HNF1? expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1?-expressing cells we used a tamoxifen-inducible Hnf1?CreER/R26R(Yfp/LacZ) mouse to lineage-trace Hnf1?(+) biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1?(+) cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1?(+) cells to hepatocyte was detected. We next assessed the contribution of Hnf1?(+) -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1?(+) cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1?(+) cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1?(+) -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways.HNF1?(+) biliary duct cells are the origin of LPC. HNF1?(+) cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration.

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Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition.

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