HNF4

Protein Interactions

When Drosophila and rat HNF4 mRNAs are co-translated and tested in a gel-shift assay, the translation products form an intermediate gel-shift band indicative of heterodimer fomation. This is further evidence of the close relationship between these two proteins (Zhong, 1993).

A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4

With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. TAP, using Protein A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein A and CBP. FLAG-His purification works sufficiently well so that two cofactors of the Drosophila nuclear receptor protein dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. The two proteins increase the ability of dHNF4 to bind DNA in vitro and to function in vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms (Yang, 2006).

Previous studies of the Drosophila Ecdysone nuclear receptor and its heterodimer partner Ultraspiracle (EcR/USP) have shown that they require Hsc70 and Hsp83 for full DNA binding activity. To see if these two chaperones act similarly to increase the DNA-binding activity of dHNF4, electrophoretic gel mobility shift assays were performed in the presence of rabbit reticulocyte lysate (RT) or purified Hsc70 and Hsp83 proteins. Reticulocyte lysate is a rich source of Hsp90 and Hsp70, mammalian Hsp83 and Hsc70 homologues, respectively. The mobility shift assays show that reticulocyte lysate and both purified proteins enhance the binding of dHNF4 to its cognate DNA element. Interestingly, the enhancing activity of the reticulocyte lysate was higher than for either Hsc70 or Hsp83 on their own. Saturation of dHNF4 DNA binding activity enhancement was reached with Hsc70 or Hsp83 amounts of about 1.2 mg per reaction. When both chaperones were combined, however, dHNF4 DNA-binding activity could be further enhanced, suggesting that the two proteins act in a complementary or synergistic fashion (Yang, 2006).

As with EcR, dHNF4 associates specifically with the Hsp83 and Hsc70 chaperone proteins, and that these play an important role in DNA binding. Interestingly, this is the first non-steroid binding nuclear receptor shown to bind these proteins. Although the natural ligand for dHNF4 is unknown, mammalian HNF4 protein expressed in bacteria has been shown to bind small fatty acids related to and including palmitate within its ligand binding domain. This is the case for dHNF4 expressed in bacteria or Sf9 cells. Chaperones may be required to stabilize dHNF4 prior to fatty acid binding, or to facilitate exchange with some other native ligand. Additional or alternative roles are the modulation of intra- or inter-molecular interactions that control DNA binding and/or transcriptional activity. Mammalian HNF4 proteins play pivotal roles in lipid metabolism and associated diseases such as diabetes and heart disease. Hence, the interactions described in this study, and their molecular and physiological effects, will be important subjects for further study (Yang, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

Hnf4 is expressed in developing Drosophila embryos in midgut, fat bodies and Malpighian tubules, a strikingly similar pattern to its limited expression in the adult intestine, liver and kidney of the mouse homolog.

Maternal mRNA is deposited in the egg by nurse cells. The highest level of Drosophila HNF4 mRNA is found in stage 1 and stage 2 embryos where the mRNA shows uniform ectodermal distributed. During cleavage stages, the mRNA is retained at the peripheral regions of the syncytial blastoderm. Just before cellularization, the only detectable stain in the syncytial blastoderm is terminal, with the posterior end being stained more strongly than the anterior. From 2 to 6 hours after fertilization, there is no detectable Drosophila HNF4 mRNA. The mRNA then reappears between 6 and 8 hours, initially in the endodermal cells corresponding to the invaginating posterior midgut primordium and later in the anterior midgut primordium. The stain grows more intense, definitely conforming to the distribution of the dividing endodermal cells in the midgut. The cells of the foregut and hindgut contain little or no HNF4 messenger RNA.

Still later (stage 14/15) a variety of tissues contain HNF4 mRNA. These include fat bodies, Malpighian tubules, salivary glands and one cluster of cells, found on either side of each of the embryonic abdominal segments; the nature of these cells is unknown but may be related to the peripheral nervous system or endocrine gland. Staining in the Malpighian tubules is confined to the distal part of each tubule. This distal region contains dividing cells that are responsible for the elongation of the Malpighian tubules. At the end of stage 15, when the fused midgut has contracted to form four loops, the most heavily stained region in the midgut is observed in the midgut caeca and in the first and fourth loops from which gut primordia (nests of imaginal cells rather than imaginal discs) arise in larvae (Zhong, 1993).

Conflicting with the results given just above is another paper, reporting that there is no indication that Drosophila Hnf4 is expressed in the fat body, and that it is not involved in the development of this tissue (Hoshizaki, 1994).

Comparison of Drosophila Hnf4 distribution with that of Forkhead, shows that Hnf4 transcription is confined to the midgut, while Forkhead is present in foregut and hindgut. Both proteins are present in the salivary gland and the Malpighian tubules (Zhong, 1993)

Effects of Mutation or Deletion

A Drosophila mutant that has a chromosome deletion spanning the Hnf4 locus fails to develop tissues where Hnf4 is expressed during late embryogenesis. Early midgut, Malpighian tubule and salivary gland development appear to be normal. However, after stage 10, when the HNF4 mRNA reappears in wild type embryos, there are clearly visible defects in midgut, Malpighian tubule and salivary gland development in the mutants. Both the anterior and posterior midgut fail to further invaginate, the malpighian tubules fail to grow and the salivary gland invagination is arrested. At around stage 16, the endodermal part of the midgut is clearly missing, the Malpighian tubules are not formed, and the salivary glands do not invaginate properly and are reduced significantly in size (Zhong, 1993)

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date revised: 20 December 2009

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