Isoforms of Hepatocyte Nuclear Factor-6 Differ in DNA-binding Properties, Contain a Bifunctional Homeodomain, and Define the New ONECUT Class of Homeodomain Proteins

Abstract

Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6␣ and HNF-6␤ for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.The phenotype of multicellular organisms is determined in part by the cell type-specific expression of genes. Since the initial observation that expression of liver-specific genes is controlled at the level of transcription (1), several liver-enriched transcription factors have been identified and extensively studied. These factors contain DNA-binding domains that have been conserved throughout evolution. Based on the structure of their functional domains, the liver-enriched transcription factors were classified into five families (2, 3). These include the CCAAT/enhancer binding proteins and the proline acid-rich factors, which both contain a leucine zipper, the homeodomain proteins of the hepatocyte nuclear factor (HNF)

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells

“…The molecular dissection of OC1 revealed that the cut domain and the homeodomain act together to determine DNA-binding affinity, specificity and transactivation. 20 The sequence conservation in these domains among the three paralogues suggests that OC2 binds a similar DNA consensus sequence to OC1. Indeed, many of the OC1 targets are also recognized by OC2.…”

“…The alignment of these sequences revealed the consensus, 5 0 -AATCG(A)ATA(C)-3 0 (Figure 4b), which overlapped significantly with those of the cut domain of human CDP (5 0 -ATCGAT-3 0 ) and OC1 (5 0 -ATCAAT-3 0 ). 20,22 Based on the consensus-binding sequence of OC2, we searched for candidate target genes encoding proteins related to Th1 function by checking the genome sequences of the promoter region. We found tandem repeats of the consensus sequence 5 0 upstream of the human T-bet gene, 5 0 -ATCAATAAAGATCGAT-3 0 , in the region from À490 to À475 (the consensus sequences are underlined and designated as 'site 1' and 'site 2', Figure S1) were mutated in the reporter containing OC2 promoter (À451 to þ 124) and the reporter activities of each construct, as well as the wild-type (top) and OC2 promoter (À347 to þ 124, bottom), were examined in the presence or absence of T-bet in COS7 cells.…”

Liver glucokinase gene expression is controlled by the onecut transcription factor hepatocyte nuclear factor-6

“…pECE-HNF6α, pECE-HNF6β and pXJ42-OC2 have been described [12,13,14]. The pXP2-GKS1 firefly luciferase reporter vector contains the hepatic promoter of the mouse glucokinase gene (-987 to +19) cloned in the SacI and BglII sites of pXP2.…”

“…This fragment includes the distal (-7613 to -7622) HNF-6 binding site. The internal control vector pRL-138 coding Renilla luciferase contains the pfk-2 promoter (-138 to +86) cloned in pRLnull (Promega, Leiden, The Netherlands) [13].…”

“…A candidate is the hepatocyte nuclear factor (HNF)-6 [12]. This transcription factor is the prototype of the ONECUT class of homeodomain proteins, the members of which possess two DNA-binding domains, a cut domain and a divergent homeodomain [13]. Two members of this class are present in mammalian liver, namely HNF-6 and Onecut-2 (OC-2) [14].…”