Crystallization of hepatocyte nuclear factor 4α (HNF4α) in complex with the HNF1α promoter element

Lü, Peng; Liu, Jianguo; Melikishvili, Manana; Fried, Mike; In, Young

doi:10.1107/s1744309108007136

Cited by 9 publications

(8 citation statements)

References 31 publications

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“…3B), consistent with the stoichiometric ratio of 2:1 found previously (36). When examined by an EMSA titration experiment, binding is significantly cooperative ( ϭ 67 Ϯ 14) despite the small dimer interface between the proteins.…”

Section: Overall Structure Of Hnf4␣-dbd and Comparison With The Relatsupporting

confidence: 88%

“…HNF4␣-DBD was released by TEV digestion from amylose magnetic beads (New England Biolabs) after overnight incubation at 4°C and further purified by ion exchange chromatography (Mono-S FPLC). The purified protein was estimated to be at least 98% pure as judged by staining with Coomassie Brilliant Blue on an 8 -25% gradient SDS-polyacrylamide gel (36). Fractions were pooled; concentrations were measured by UV absorption and stored at Ϫ80°C as a 10% (v/v) glycerol stock.…”

Section: Construction Of Overexpression Vectors For Wild Type Andmentioning

confidence: 99%

“…Crystallization and Data Collection-Crystallization of the complex has been reported previously (36). Briefly, protein-DNA complexes were made by dialyzing them with a molar ratio of 2 HNF4␣ and 1.2 double-stranded DNA in 20 mM Tris, pH 7.5, 75 mM NaCl, and 1 mM dithiothreitol at 4°C for 2.5 h and concentrated to at least 10 mg ml…”

Section: Construction Of Overexpression Vectors For Wild Type Andmentioning

confidence: 99%

“…Elements-The crystals of HNF4␣-(46 -126), containing two zinc finger motifs and the C-terminal extension "T-box" in complex with DNA were prepared as previously reported (36), and the structure was determined by conventional methods as described under "Experimental Procedures." Fig.…”

Section: Overall Structure Of Hnf4␣-dbd and Comparison With The Relatmentioning

confidence: 99%

“…DNA concentrations were measured by absorbance, using a molar extinction coefficient (⑀ 260 1 cm ϭ 2.57 ϫ 10 5 ). Pure recombinant wild type HNF4␣-DBD-(46 -126) was prepared as described previously (36). Samples were stored at Ϫ20°C until use.…”

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confidence: 99%

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Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4α

Lü

Rha

Melikishvili

et al. 2008

Journal of Biological Chemistry

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HNF4␣ (hepatocyte nuclear factor 4␣) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic ␤-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4␣ is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4␣ recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 Å crystal structure of human HNF4␣ DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1␣, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4␣ molecular function can cause significant effects in afflicted MODY patients.HNF4␣ is a novel member (NR2A1) of the nuclear receptor (NR) 2 family (1) and plays a crucial role in the development and function of vital organs. For example, HNF4␣ is essential for development of the liver (2), colon (3), and pancreas (4), and deletion of the HNF4␣ gene from the mouse genome results in embryonic lethality due to the failure to undergo normal gastrulation (5, 6). Conditional targeted gene disruption of HFN4␣ results in marked metabolic disregulation and increased mortality (7-9). In addition, HNF4␣ is known to activate a wide variety of genes involved in glucose, fatty acid, cholesterol, and amino acid metabolism in the liver, kidney, intestine, and pancreas (10 -12), including counterpart transcription factors, such as HNF1␣ (13), HNF6 (14), and pregnane X receptor (15), that in turn control additional liver and ␤-cell-specific target genes. Further underscoring the importance of HNF4␣ in pancreatic ␤-cells, mutations on HNF4␣ in humans are directly linked to the onset of MODY1 (maturity onset diabetes of the young 1) (Fig. 1A), one of the most common monogenic causes of diabetes, mainly characterized by impairment of glucosestimulated insulin secretion from the ␤-cells (16). A direct link between HNF4␣ and glucose-stimulated insulin secretion by pancreatic ␤-cells has been proven by cellular studies (17,18) and pancreatic ␤-cell-specific HNF4␣ knock-out mice (17). A recent genome-wide expression profiling study revealed that both HNF1␣ and HNF4␣ are the master regulators of the ␤-cells directly affecting their physiology (19,20). HNF4␣ is functionally very closely related to HNF1␣, since they crossregulate each other and form a common network of transcription factors that controls the development and function of hepatocyte and pancreatic islets (21, 22). A...

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Section: Overall Structure Of Hnf4␣-dbd and Comparison With The Relatsupporting

confidence: 88%

Section: Construction Of Overexpression Vectors For Wild Type Andmentioning

confidence: 99%

Section: Construction Of Overexpression Vectors For Wild Type Andmentioning

confidence: 99%

Section: Overall Structure Of Hnf4␣-dbd and Comparison With The Relatmentioning

confidence: 99%

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confidence: 99%

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