1H, 13C and 15N backbone resonance assignments of hepatocyte nuclear factor-1-beta (HNF1ß) POUS and POUHD.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1ß plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1ß POUS and POUHD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1ß cause diseases.

Role of transcription factor hepatocyte nuclear factor-1ß in polycystic kidney disease.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a DNA-binding transcription factor that is essential for normal kidney development. Mutations of HNF1B in humans produce cystic kidney diseases, including renal cysts and diabetes, multicystic dysplastic kidneys, glomerulocystic kidney disease, and autosomal dominant tubulointerstitial kidney disease. Expression of HNF1B is reduced in cystic kidneys from humans with ADPKD, and HNF1B has been identified as a modifier gene in PKD. Genome-wide analysis of chromatin binding has revealed that HNF-1ß directly regulates the expression of known PKD genes, such as PKHD1 and PKD2, as well as genes involved in PKD pathogenesis, including cAMP-dependent signaling, renal fibrosis, and Wnt signaling. In addition, a role of HNF-1ß in regulating the expression of noncoding RNAs (microRNAs and long noncoding RNAs) has been identified. These findings indicate that HNF-1ß regulates a transcriptional and post-transcriptional network that plays a central role in renal cystogenesis.

Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

Elucidating the Mutational Landscape in Hepatocyte Nuclear Factor 1ß (HNF1B) by Computational Approach.

Transcription factors are the major gene-regulatory proteins that recognize specific nucleotide sequences and bind to them. Missense mutations in transcription factors play a significant role in misregulation of gene expression contributing to various diseases and disorders. Understanding their structural and functional impact of the disease-causing mutations becomes prime importance in treating a disease. Commonly associated defect with the mutations of hepatocyte nuclear factor 1 beta (HNF1B) protein, a transcription factor results in maturity-onset diabetes of the young-5 (MODY-5) leading to loss of function. In the study presented, we applied a series of computational strategies to analyze the effect of mutations on protein structure or function in protein-DNA complex. The mutations from publicly available databases were retrieved and subjected to an array of in silico prediction methods. Key implementation of the present study consists of a pipeline drawn using well established in silico prediction methods of different algorithms to explain the biochemical changes impaired upon mutations in the binding sites of protein-DNA complex using HNF1B. Prediction scores obtained from the in silico tools suggested H153N and A241T as the major nsSNPs involved in destabilizing the protein. Further, high-end microscopic computational study, such as molecular dynamics simulations was utilized to relate the structural and functional effects upon mutations. Although, both the mutations exhibited similar structural differences, we observed A241T with higher destabilizing effect on the protein. The presented work is a step toward understanding the genotype-phenotype relationships in transcription factor genes using fast and accurate computational approach.

Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1ß (HNF1ß) transcription factor is imported into the nucleus via a monopartite NLS sequence.

The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1ß transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1ß and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1ß interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1ß (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1ß NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1ß and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1ß via its nuclear import pathway.

Human mutations affect the epigenetic/bookmarking function of HNF1B.

Bookmarking factors are transcriptional regulators involved in the mitotic transmission of epigenetic information via their ability to remain associated with mitotic chromatin. The mechanisms through which bookmarking factors bind to mitotic chromatin remain poorly understood. HNF1ß is a bookmarking transcription factor that is frequently mutated in patients suffering from renal multicystic dysplasia and diabetes. Here, we show that HNF1ß bookmarking activity is impaired by naturally occurring mutations found in patients. Interestingly, this defect in HNF1ß mitotic chromatin association is rescued by an abrupt decrease in temperature. The rapid relocalization to mitotic chromatin is reversible and driven by a specific switch in DNA-binding ability of HNF1ß mutants. Furthermore, we demonstrate that importin-ß is involved in the maintenance of the mitotic retention of HNF1ß, suggesting a functional link between the nuclear import system and the mitotic localization/translocation of bookmarking factors. Altogether, our studies have disclosed novel aspects on the mechanisms and the genetic programs that account for the mitotic association of HNF1ß, a bookmarking factor that plays crucial roles in the epigenetic transmission of information through the cell cycle.

Structural basis of disease-causing mutations in hepatocyte nuclear factor 1beta.

HNF1beta 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 HNF1beta 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 HNF1beta 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 HNF1alpha, 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.

Crystallization of hepatocyte nuclear factor 1beta in complex with DNA.

Hepatocyte nuclear factor 1beta (HNF1beta) is a member of the POU transcription-factor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1beta-DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2 A (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43 A. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1beta and the molecular basis of the disease-causing mutations found in it.

Mutations in hepatocyte nuclear factor-1beta and their related phenotypes.

BACKGROUND: Hepatocyte nuclear factor-1 beta (HNF-1beta) is a widely distributed transcription factor which plays a critical role in embryonic development of the kidney, pancreas, liver, and Mullerian duct. Thirty HNF-1beta mutations have been reported in patients with renal cysts and other renal developmental disorders, young-onset diabetes, pancreatic atrophy, abnormal liver function tests, and genital tract abnormalities. METHODS: We sequenced the HNF-1beta gene in 160 unrelated subjects with renal disease, 40% of whom had a personal/family history of diabetes. RESULTS: Twenty three different heterozygous HNF-1beta mutations were identified in 23/160 subjects (14%), including 10 novel mutations (V61G, V110G, S148L, K156E, Q176X, R276Q, S281fsinsC, R295P, H324fsdelCA, Q470X). Seven (30%) cases were proven to be due to de novo mutations. Renal cysts were found in 19/23 (83%) patients (four with glomerulocystic kidney disease, GCKD) and diabetes in 11/23 (48%, while three other families had a family history of diabetes. Only 26% of families met diagnostic criteria for maturity-onset diabetes of the young (MODY) but 39% had renal cysts and diabetes (RCAD). We found no clear genotype/phenotype relationships. CONCLUSION: We report the largest series to date of HNF-1beta mutations and confirm HNF-1beta mutations as an important cause of renal disease. Despite the original description of HNF-1beta as a MODY gene, a personal/family history of diabetes is often absent and the most common clinical manifestation is renal cysts. Molecular genetic testing for HNF-1beta mutations should be considered in patients with unexplained renal cysts (including GCKD), especially when associated with diabetes, early-onset gout, or uterine abnormalities.