Information about HNF4

Protein Purification and Cloning
Hepatocyte nuclear factor 4 (HNF4, HNF-4) was originally identified as an activity in crude rat liver nuclear extracts that bound DNA elements required for the transcription of two liver-specific genes -- transthyretin (TTR) and apolipoprotein CIII (apoCIII). After protein purification and cloning, it was found that HNF4 is a member of the nuclear receptor superfamily (NR2A1) of ligand-dependent transcription factors (Sladek et al., 1990). However, since a ligand has not yet been definitively identified for HNF4, it is currently referred to as an orphan receptor. HNF4 is highly conserved from species ranging from insect to man.


Figure 1.
Amino acid conservation of HNF4a genes across species and in comparison to other HNF4 genes and the closest relative in the nuclear receptor superfamily, RXR. %, % amino acid identity to human HNF4a1. Zn++, zinc finger region/DNA binding; Ligand?, putative ligand binding domain. Numbering from an alternative start site of translation is shown in parentheses. A-F, conventional nomenclature for nuclear receptor domains.

HNF4a gene and Splicing Variations
The first HNF4 gene identified is now called HNF4a and is located on human chromosome 20 q13.1-13.2. The gene consists of at least 12 exons and spans 30 kb. Two related genes, HNF4b and HNF4g have also been identified but much less is known about those genes. Splicing variations in the N- and C-terminus of the HNF4a gene may yield as many as 9 different HNF4a isoforms. The significance of these different isoforms is not well understood but they appear to exhibit slightly different function and tissue distribution.


Figure 2.
Structure of the human HNF4a gene and the isoforms generated by alternative splicing events.

Tissue Distribution and Role in vivo
In addition to a relatively high level of expression in the liver, HNF4a mRNA and protein is also found in the kidney, intestine and colon and to a lesser extent pancreas and stomach. HNF4a is expressed early in development, visible by in situ hybridization in the mouse visceral endoderm at embryonic day 4.5, long before liver development. Mice deleted in the HNF4a gene die in utero between day 9.5 and 10.5 and exhibit impaired gastrulation. Whereas HNF4a appears to be essential in the visceral endoderm it may not be necessary for the earliest steps in the development of the fetal liver (Li et al., 2000 ). However, in the adult liver, HNF4a is required for proper liver function (Hayhurst et al., 2001 ).

HNF4a Target Genes and Human Disease
Over 55 distinct target genes have been identified for HNF4a. Since many of those genes contain more than one HNF4a binding site, the total number of distinct, non species redundant HNF4a binding sites is now 74. These genes can be grouped into several different categories, according to function, such as nutrient transport and metabolism, blood maintenance, immune function, liver differentiation and growth factors. The best characterized HNF4a target genes are those involved in lipid transport (e.g., apolipoprotein genes) and glucose metabolism (e.g., L-PK and PEPCK). Nearly all of the target genes identified thus far are expressed primarily in the liver; several are expressed in other organs as well, such as the pancreas. Even though there is quite a lot of HNF4a protein in the kidney, only one kidney specific target has been identified thus far - erthyropoietin (Epo). HNF4a activates the Epo gene in concert with hypoxia inducible factor (HIF) in response to hypoxic conditions.

Adobe Acrobat File of Target Genes
Figure 3.
Listed are HNF4a target genes grouped by function. All genes contain HNF4a binding sites that have been verified by one or more methods. Since HNF4a binding sites are highly conserved between species, only one species for each gene is shown - human (h) or the species most closely related to human (r, rat; m, mouse; rab, rabbit, b, bovine; w, woodchuck; Xn, Xenopus laevis; mit, mitochondrial). Consensus sequence: Upper case letters, nucleotides that appear in that position 25% or more of sites; Lower case letters, nucleotides that appear in that position in 10 to 25% of the sites. Note that this is a very approximate consensus sequence that changes as more sites are added to the list.

Most HNF4a binding sites in the target genes can be considered as direct repeats of AGGTCA with a spacing of one nucleotide (e.g., AGGTCAxAGGTCA). HNF4a, however, also binds sites separated by 2 nucleotides (DR2) but not 0, 3, 4 or 5 nucleotides to any appreciably degree. Whereas there can be considerable variation from the consensus AGGTCA in either half site, the HNF4a binding sites can often be identified by three A's in the middle of the site.

Mutations in HNF4a binding sites have been linked directly to human diseases, most notably hemophilia and maturity onset diabetes of the young (MODY) 1. For example, several mutations in HNF4a binding sites have been found in the promoter of blood coagulation Factor VII and Factor IX genes, resulting in hemophilia, and HNF1a and HNF4a genes, resulting in MODY. Through other target genes, HNF4a is potentially linked to other diseases such as atherosclerosis and cancer (Hepatocyte nuclear factor (HNF)4a and human disease).

Mutations have also been found in the HNF4a gene itself. Many of these are linked to MODY1 but several others are currently classified as polymorphisms as it is not yet clear whether they affect HNF4a function.


Figure 4.
Shown are mutations found in the HNF4a coding sequence associated with Maturity Onset Diabetes of the Young 1 (MODY1). Given is the codon number and amino acid in single letter code. X, stop codon,; fs, frameshift; del, deletion; ins, insertion ( ), association with MODY1 is not certain, may be polymorphisms. Zn++, zinc finger/DNA binding domain; Ligand?, putative ligand binding domain. Not shown are mutations found in the HNF4a gene promoter associated with MODY1. Note, only heterozygous mutations have been identified.

Classification of HNF4 as a Novel Nuclear Receptor
HNF4a is rather unique in that it binds DNA exclusively as a homodimer and yet is localized primarily in the nucleus and binds only DNA response elements consisting of direct repeats (Nuclear Receptor Subfamilies). It also activates transcription and binds several different co-activators (such as GRIP1, SRC, p300/CBP, DRIP205) in the absence of exogenously added ligand. Fatty acyl Co-enzyme A thioesters were proposed as ligands for HNF4a in 1998 but those compounds have not been found to affect binding of co-activator or co-repressor in vitro so they do not appear to act as traditional ligands.


Figure 5.
Zinc++, Zinc finger region; Ligand?, region highly similar to the ligand binding domain of other nuclear receptors. H9, H10, helix 9 and Helix 10. AF-1 and AF-2, Activation function 1 and 2. Many co-activators bind the AF-2, including p300/CBP, p160 proteins and the DRIP complex. Co-activators binding to AF-1 are indicated.

HNF4 genes also possess a pair of charged residues in helices 9 and 10 in the putative ligand binding domain that appear to be critical for homodimerization and to play a role in preventing heterodimerization with other receptors such as RXR (Bogan et al., 2000). This pair of residues can be used as a signature for membership in the HNF4 subfamily of genes as all bonafide HNF4 genes to date have this unique pair of charged residues.


Figure 6.
Alignment of the amino acids in the region of helix 9 and 10 of all identified HNF4 genes from the species indicated. Dash, amino acids identical to human. The C. elegans HNF4-like protein identified in 1994 does not contain the signature K(X)₂₆E/D motif.

The above information is adapted from a book chapter written by Sladek and Seidel, 2001. Only the most critical references and those not included in that review are cited here.