Crystallization of hepatocyte nuclear factor 1β in complex with DNA

“…Preparation of the samples and crystallization of the complex have been reported previously (31). The crystals belong to the space group R3 with unit cell dimensions a = b = 174.69 Å and c = 72.43 Å and diffraction to 3.2 Å resolution.…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

“…Electrophoresis continued for about an hour before the gel was stained with 0.1 μg/mL ethidium bromide in 0.5× TBE buffer. For these experiments, we used the same oligo (21-mer overhang) that was used for crystallization and structure determination (31).…”

Section: Electromobility Shift Assaymentioning

confidence: 99%

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Structural Basis of Disease-Causing Mutations in Hepatocyte Nuclear Factor 1β^,

2007

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HNF1β is an atypical POU transcription factor that participates in a hierarchical network of transcription factors controlling the development and proper function of vital organs such as liver, pancreas, and kidney. Many inheritable mutations on HNF1β are the monogenic causes of diabetes and several kidney diseases. To elucidate the molecular mechanism of its function and the structural basis of mutations, we have determined the crystal structure of human HNF1β DNA binding domain in complex with a high-affinity promoter. Disease-causing mutations have been mapped to our structure, and their predicted effects have been tested by a set of biochemical/ functional studies. These findings together with earlier findings with a homologous protein HNF1α, help us to understand the structural basis of promoter recognition by these atypical POU transcription factors and the site-specific functional disruption by disease-causing mutations.HNF1β (hepatocyte nuclear factor 1β; also known as vHNF1 or TCF2) is a widely distributed transcription factor that plays a critical role in early vertebrate development and embryonic survival (1-3). First identified as a key regulator in the liver, HNF1β is also expressed in the pancreas, kidney, lung, ovary, testis, and throughout the gastrointestinal tract. In pancreatic β-cells, HNF1β is known to form an integrated regulatory network with other transcription factors such as HNF1α, HNF4α, Pdx-1, Foxa2, and NeuroD1 for organ development and proper function (1,4). Thus, in humans, heterozygous mutations in the HNF1β gene have been linked to neonatal diabetes (5) and the autosomal dominant subtype of diabetes known as MODY (maturity-onset diabetes of the young) (6). Extrapancreatic diseases are also increasingly recognized in different organs, especially in the kidney, with a variety of renal developmental disorders such as renal cysts, familial hypoplastic glomerulocystic kidney disease, renal malformation, and atypical familial hyperuricaemic nephropathy (7-11).POU transcription factors, which include Pit-1, Oct-1, and Unc-86 as founding members and are now expanded to more than 13 members in humans, are developmental regulators of various neuroendocrine organs, and their sequence-specific DNA binding is mediated by a bipartite motif that consists of a POU homeodomain (POU H ) and POU-specific domain (POU S ) (12,13). POU H is a 60 amino acid classic homeodomain made of three α-helices with the third as a DNA recognition helix, while POU S is an additional ∼75 amino acid all-α-helical motif that cooperates with POU H to enhance the binding affinity and specificity of DNA binding ( Figures 1 and 2) (12,14). HNF1α (MODY3 gene product, the most commonly mutated MODY protein) and HNF1β (MODY5 gene product) are atypical members of the POU transcription factors. Their POU S domains have at least one additional α-helix at the N-terminus, and the second † This work was funded by the Juvenile Diabetes Research Foundation (1-2004-506) (8,24). Recently, HNF1β has also been associated wi...

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“…no. P2187) was carried out in binding buffer (20% glycerol, 5 mM MgCl 2 , 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5 mg/ml BSA), in the presence of 10 nM estradiol at room temperature for 30 minutes, as described previously (Lu et al, 2006). Samples were resolved on 6% native polyacrylamide gel electrophoresis (PAGE) and stained with ethidium bromide (EtBr).…”

Section: Ere Scans and Gel Retardation Assaysmentioning

confidence: 99%

Lkb1inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy

Contreras¹,

Akbay²,

Gallardo³

et al. 2010

Disease Models &Amp; Mechanisms

105

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SUMMARYEndometrial cancer -the most common malignancy of the female reproductive tract -arises from the specialized epithelial cells that line the inner surface of the uterus. Although significant advances have been made in our understanding of this disease in recent years, one significant limitation has been the lack of a diverse genetic toolkit for the generation of mouse models. We identified a novel endometrial-specific gene, Sprr2f, and developed a Sprr2f-Cre transgene for conditional gene targeting within endometrial epithelium. We then used this tool to generate a completely penetrant Lkb1 (also known as Stk11)-based mouse model of invasive endometrial cancer. Strikingly, female mice with homozygous endometrial Lkb1 inactivation did not harbor discrete endometrial neoplasms, but instead underwent diffuse malignant transformation of their entire endometrium with rapid extrauterine spread and death, suggesting that Lkb1 inactivation was sufficient to promote the development of invasive endometrial cancer. Mice with heterozygous endometrial Lkb1 inactivation only rarely developed tumors, which were focal and arose with much longer latency, arguing against the idea -suggested by some prior studies -that Lkb1 is a haploinsufficient tumor suppressor. Lastly, the finding that endometrial cancer cell lines were especially sensitive to the mTOR (mammalian target of rapamycin) inhibitor rapamycin prompted us to test its efficacy against Lkb1-driven endometrial cancers. Rapamycin monotherapy not only greatly slowed disease progression, but also led to striking regression of pre-existing tumors. These studies demonstrate that Lkb1 is a uniquely potent endometrial tumor suppressor, but also suggest that the clinical responses of some types of invasive cancers to mTOR inhibitors may be linked to Lkb1 status.

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Crystallization of hepatocyte nuclear factor 1β in complex with DNA

Cited by 7 publications

References 26 publications

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Structural Basis of Disease-Causing Mutations in Hepatocyte Nuclear Factor 1β^,

Lkb1inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy

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Crystallization of hepatocyte nuclear factor 1β in complex with DNA

Cited by 7 publications

References 26 publications

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Structural Basis of Disease-Causing Mutations in Hepatocyte Nuclear Factor 1β,

Lkb1inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy

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Product

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Structural Basis of Disease-Causing Mutations in Hepatocyte Nuclear Factor 1β^,