Cloning And Expression Of HNF-3-1,HNF-3-2 In Amphioxus Branchiostoma Belcheri Tsingtauense

Hepatocyte nuclear factors(HNFs) play important roles in regulating transcription of the liver-sepcific genes.These families include the family HNF-1,the leucine zipper C/EBP(CCAAT/enhancer bingding protein) family,the HNF-3 winged helix family, and the families HNF-4,DBP,HNF-6.They are mainly expressed in the metabolic organs including liver and pancreas etc of vertebrate.HNFs form a complicated hierarchical network regulating liver development and differentiation.Hepatocyte nuclear factor 3(HNF-3) proteins play pivotal roles in the regulation of metabolism and the differentiation of the metabolic tissues such as pancreas and liver.Amphioxus or lancelet,a cephalochordate,has long been regarded as the closest relative of vertebrates,and is becoming an emerging model organism for insights into the origin and evolution of vertebrates.We isolated two gene fragments exhibiting identity to hepatocyte nuclear factor 3-1,3-2 genes.The purposes of this study were thus to examine the expression pattern of the two genes in adult amphioxus,to test their expression regulation in response to challenge with exogenous recombinant growth hormone(GH) and to assay the role of BbHNF-3-1,3-2 in GH regulation of gene expression in the liver in vivo.Frist,we cloned the amphioxus BbHNF-3-1 cDNA with an ORF coding for a protein of 404 amino acids and BbHNF-3-2 cDNA with an ORF coding for a protein of 445 amino acids respectively.Both of the two ORFs contain a conserved 100 amino acid DNA-binding domain,which is centrally located in the HNF-3.This DNA-binding domain is made up of three a-helices and two characteristic large loops,or 'wings', resulting in the designation of this DNA-binding motif as the 'winged helix DNA-binding domain'.This domain can bind to unique DNA sequences in the promoter and enhancer regions of genes expressed in terminaly differented hepatocytes.HNF-3 is mainly expressed in liver of vertebrate,and also at a low level in spleen, kidney,heart,gut,lung,brain etc.It is to be believed that the hepatic caecum in amphioxus is homologous to the vertebrate liver.Northern blotting revealed that Bb HNF-3 transcript was most abundant in the hepatic caecum,and also presented in the hind-gut and gill,while it was absent in the muscle,testis,ovary,neural tube and notochord.This basically agrees with the notion that the hepatic caecum is the precursor of vertebrate liver.Challenge with GH soon resulted in an up-regulation of BbHNF-3-2 expression in the guts including the hepatic caecum.In contrast,little BbHNF-3-1 expression change was observed in the hepatic caecum following GH challenge.Cloning and sequencing the promoter region of this gene revealed two binding elements for signal transducers and activators of transcription 5(STAT5),suggesting that GH regulation of BbHNF-3-2 expression in the liver may be mediated through direct binding of STAT5 to the BbHNF-3-2 promoter,which is similar with the vertebrate.