The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids¹

Abstract

The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer. HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding. Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homote tramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids. Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human α1-antitrypsin TATA box region. Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH. Interestingly, DCoH retains its enzymatic activity while complexed with HNF1. Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

Introduction

The regulation of liver-specific gene expression is brought about mainly at the transcriptional level (Derman et al., 1981) by “liver-enriched” transcription factors. The various forms of the hepatocyte nuclear factor-1 (HNF1) constitute a family of homeodomain containing factors important for tissue-specific regulation of transcription Tronche and Yaniv 1992, Tronche et al 1994. HNF1α and HNF1β (or v-HNF1) are homologous proteins that bind their target DNA as homo- or heterodimers. The dimerisation and DNA binding domains are confined to the N-terminal 281 residues which are shared by all family members. Sequence-specific DNA binding is achieved through two domains, an atypical homeodomain that is 21 residues larger than classical ones and a domain related to the POU-specific A-box Rosenfeld 1991, Ceska et al 1993. Tomei and co-workers showed that the dimerisation domain increased DNA binding affinity (Tomei et al., 1992). The dimerisation domain which shows homology to sequences of the myosin heavy chain Chouard et al 1990, Nicosia et al 1990, was proposed to form an extended chain segment followed by two helices Pastore et al 1991, Pastore et al 1992.

HNF1 dimers can associate with two molecules of the “dimerization co-factor of HNF1” (DCoH) to form stable heterotetramers. DCoH is a bifunctional protein, also known as PCD (pterin-4a-carbinolamine dehydratase). A homotetramer of DCoH has an enzymatic activity involved in the aromatic amino acids hydroxylation pathway contributing to the tetrahydrobiopterin (BH4) regeneration (Citron et al., 1992) while the dimeric form of DCoH stabilizes HNF1 dimers by binding to its dimerization domain (Mendel et al., 1991), presumably forming a four-helix bundle (Ficner et al., 1995). Although DCoH itself fails to bind DNA and does not activate transcription when fused to a DNA binding domain, it substantially enhances transcriptional activation by HNF1 (Mendel et al., 1991).

The three-dimensional structure of the dimericunit of DCoH Ficner et al 1995, Endrizzi et al 1995 is reminiscent of the saddle-like structure of the TATA-box binding protein (TBP) Nikolov et al 1992, Kim et al 1993. The function of its concave, hydrophobic surface remains elusive but presumably provides a docking site for other macromolecules (Ficner et al., 1995). It is likely that DCoH interacts with other ligands apart from HNF1 because it also functions in the absence of HNF1, e.g. during early developmental stages of Xenopus(Pogge von Strandmann & Ryffel, 1995). Accumulating evidence suggests a broader role of DCoH in the regulation of transcription than was suspected initially (Hansen & Crabtree, 1993): the bacterial homologue phhB is apparently required for the expression of phenylalanine hydroxylase in Pseudomonas aeruginosa(Zhao et al., 1994).

In this study we utilized purified, recombinant HNF1, HNF1/DCoH and DCoH in order to evaluate how protein-protein interactions modulate their function. We confirmed that DCoH retains its enzymatic activity while in complex with HNF1. Analysis of thermal protein complex stability by temperature gradient gel electrophoresis verified the role of DCoH in stabilizing HNF1 complexes. Solid-phase footprinting Sandaltzopoulos and Becker 1994, Sandaltzopoulos et al 1995 methodology, which enables the quick and non-disruptive purification of protein/DNA complexes, allowed us to determine the stability of HNF1 or HNF1/DCoH complexes with DNA. We found that DCoH enhanced the stability of HNF1/DNA complexes and also affected the sequence specificity of DNA binding. Surprisingly, we observed RNA binding activity of HNF1 which was completely abolished in the presence of DCoH. Our results suggest that DCoH may regulate HNF1 function by modulating its interactions with nucleic acids.