The Human HNF-3 Genes: Cloning, Partial Sequence and Mutation Screening in Patients with Impaired Glucose Homeostasis

Navas, María-Ángeles; Vaisse, Christian; Boger, Stephanie; Heimesaat, Markus M.; Kollée, L.A.A.; Stoffel, Markus

doi:10.1159/000022943

Cited by 22 publications

(20 citation statements)

References 39 publications

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“…Human embryonic stem cells (hESCs) lines: H9 (WA-09, passages 51-80), ES3 (CMR[B], passages [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54], and ES5 (CMR [B], passages 27-55) and a human iPS cell line derived from a healthy individual in our laboratory, iPSC C1 (BP-143) (supplemental online Fig. 1) (passages 7-36), were maintained on irradiated human foreskin fibroblasts (HFF1, SCRC-1041; American Type Culture Collection [ATCC], Manassas, VA, http://www.atcc.org) in human embryonic stem (hES) medium composed of knockout Dulbecco's modified Eagle's medium (DMEM) (10829-018; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), supplemented with 2 mM L-GlutaMAX (35050038; Invitrogen), 50 mM b-mercaptoethanol (31350-010; Invitrogen), 13 nonessential amino acids (M7145; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), 20% knockout serum (KSR; Invitrogen), 50 U/ml penicillin, 50 mg/ml streptomycin, and 10 ng/ml of fibroblast growth factor (FGF) 2 (100-18B; PeproTech).…”

Section: Human Es Cell Propagationmentioning

confidence: 99%

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Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells

Águila¹,

Blak

Arensbergen

et al. 2014

Stem Cells Translational Medicine

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Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor that is expressed in the floor plate domain that gives rise to dopamine neurons during embryogenesis. We first validated the specificity of the vectors in human cell lines against a promoterless construct. We then selected FOXA2-positive neural progenitors from several human pluripotent stem cell lines, which demonstrated a gene expression profile typical for the ventral domain of the midbrain and floor plate, but failed to enrich for dopamine neurons. To investigate whether this was due to the selection approach, we overexpressed FOXA2 in neural progenitors derived from human pluripotent stem cell lines. FOXA2 forced expression resulted in an increased expression of floor plate but not mature neuronal markers. Furthermore, selection of the FOXA2 overexpressing fraction also failed to enrich for dopamine neurons. Collectively, our results suggest that FOXA2 is not sufficient to induce a dopaminergic fate in this system. On the other hand, our study demonstrates that a combined approach of promoter activation and lentivirus vector technology can be used as a versatile tool for the selection of a defined cell population from a variety of human pluripotent stem cell lines. STEM CELLS

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Section: Human Es Cell Propagationmentioning

confidence: 99%

“…To drive gene expression from the FOXA2 promoter, we used a cloned 59 fragment that contains the 400-bp putative promoter region [46] and generated lentiviral vectors expressing the GFP cDNA [39] controlled by this promoter sequence, pFOXA2.GFP ( Fig. 2A).…”

Section: Design and Validation Of Lentiviral Vectorsmentioning

confidence: 99%

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells

Águila¹,

Blak

Arensbergen

et al. 2014

Stem Cells Translational Medicine

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“…AH005826) was done using previously described primer sequences [22]. Single-strand conformation polymorphism (SSCP) analysis and sequencing were performed as described [19,23].…”

Section: Subjectsmentioning

confidence: 99%

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

et al. 2006

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Aims/hypothesis Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. Materials and methods Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. Results We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. Conclusions/interpretation Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

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“…Antisense and Double-stranded Oligodeoxynucleotides Studies-HNF3 antisense oligodeoxynucleotide (5Ј-CGGCTCGTGCCCTTCCAT-CTTCAC-3Ј), corresponding to codons 5-12 of human HNF3␤ mRNA (25), and NF1 antisense oligodeoxynucleotide (5Ј-CAGTGCCTCGATG-AACGGGTGGAA-3Ј), complementary to codons 4 -11 of human NF1 mRNA (26), were designed to block synthesis of these proteins. The HNF3 and NF1 sense oligodeoxynucleotides were used as controls.…”

Section: Methodsmentioning

confidence: 99%

“…To detect ALAS mRNA, a 26-mer oligodeoxynucleotide was synthesized complementary to bases ϩ328 to ϩ353 of human hepatic ALAS mRNA (29). To detect HNF3 mRNA, a 24-mer oligodeoxynucleotide was synthesized complementary to bases ϩ13 to ϩ36 of human HNF3 mRNA (25). The oligodeoxynucleotides were 5Ј-end-labeled using [␥-32 P]ATP and T4 polynucleotide kinase.…”

Section: Methodsmentioning

confidence: 99%

Hepatic Nuclear Factor 3 and Nuclear Factor 1 Regulate 5-Aminolevulinate Synthase Gene Expression and Are Involved in Insulin Repression

Scassa

Guberman

Ceruti

et al. 2004

Journal of Biological Chemistry

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Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5 -regulatory region allowed us to identify an insulin-responsive region located at ؊459 to ؊354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3␤ or HNF3␤ plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3␤ is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3␤ can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3␤ binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

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The Human HNF-3 Genes: Cloning, Partial Sequence and Mutation Screening in Patients with Impaired Glucose Homeostasis

Cited by 22 publications

References 39 publications

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

Hepatic Nuclear Factor 3 and Nuclear Factor 1 Regulate 5-Aminolevulinate Synthase Gene Expression and Are Involved in Insulin Repression

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