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1.  Ins1Cre knock-in mice for beta cell-specific gene recombination 
Diabetologia  2014;58:558-565.
Aims/hypothesis
Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain.
Methods
We describe the generation of Ins1Cre and Ins1CreERT2 mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene.
Results
We show that Ins1Cre mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1CreERT2 mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells.
Conclusions/interpretation
These two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3468-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
doi:10.1007/s00125-014-3468-5
PMCID: PMC4320308  PMID: 25500700
Beta cells; Cre recombinase; Glucose homeostasis; Hypothalamus; Insulin; Pancreatic islets; Transgenic mice
2.  Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis 
Nature genetics  2013;46(1):61-64.
The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole genome sequencing can identify all non-coding variants, yet discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in hESC-derived embryonic pancreatic progenitor cells to guide the interpretation of whole genome sequences from patients with isolated pancreatic agenesis. This uncovered six different recessive mutations in a previously uncharacterized ~400bp sequence located 25kb downstream of PTF1A (pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can uncover novel non-coding elements underlying human development and disease.
doi:10.1038/ng.2826
PMCID: PMC4131753  PMID: 24212882
3.  Pancreatic islet enhancer clusters enriched in type 2 diabetes risk–associated variants 
Nature genetics  2014;46(2):136-143.
Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central for type 2 diabetes pathogenesis, and therefore understanding islet genome regulation could provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity, and show that most such sequences reside in clusters of enhancers that form physical 3D chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers, and identify trait-associated variants that disrupt DNA-binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome, and provide systematic evidence that dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.
doi:10.1038/ng.2870
PMCID: PMC3935450  PMID: 24413736
4.  Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice 
Nature biotechnology  2013;32(1):76-83.
Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose-responsive, and reinstate normal glycemic control for up to 248 days. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3) expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta cell reprogramming through transient cytokine exposure rather than genetic modification.
doi:10.1038/nbt.2747
PMCID: PMC4096987  PMID: 24240391
5.  GATA6 haploinsufficiency causes pancreatic agenesis in humans 
Nature genetics  2011;44(1):20-22.
Understanding the regulation of pancreatic development is key for efforts to develop new regenerative therapeutic approaches for diabetes. Rare mutations in PDX1 and PTF1A can cause pancreatic agenesis, however, most instances of this disorder are of unknown origin. We report de novo heterozygous inactivating mutations in GATA6 in 15/27 (56%) individuals with pancreatic agenesis. These findings define the most common cause of human pancreatic agenesis and establish a key role for the transcription factor GATA6 in human pancreatic development.
doi:10.1038/ng.1035
PMCID: PMC4062962  PMID: 22158542
6.  Pdx1 deficiency causes mitochondrial dysfunction and defective insulin secretion through TFAM suppression 
Cell metabolism  2009;10(2):110-118.
Mutations in the transcription factor Pdx1 cause maturity-onset diabetes of the young 4 (MODY4). Islet transduction with dominant negative Pdx1 (RIPDN79PDX1) impairs mitochondrial metabolism and glucose-stimulated insulin secretion (GSIS). Transcript profiling revealed suppression of nuclear encoded mitochondrial factor A (TFAM). Herein, we show that Pdx1 suppression in adult mice reduces islet TFAM expression coinciding with hyperglycaemia. We define TFAM as a direct target of Pdx1 both in rat INS1 cells and human islets. Adenoviral overexpression of TFAM along with RIPDN79PDX1 in isolated rat islets rescued mitochondrial DNA (mtDNA) copy number and restored respiratory chain activity as well as glucose-induced ATP synthesis and insulin secretion. CGP37157, which blocks the mitochondrial Na+/Ca2+ exchanger, restored ATP generation and GSIS in RIPDN79PDX1 islets, thereby bypassing the transcriptional defect. Thus, the genetic control by the β-cell specific factor Pdx1 of the ubiquitous gene TFAM maintains β-cell mtDNA vital for ATP production and normal GSIS.
doi:10.1016/j.cmet.2009.07.002
PMCID: PMC4012862  PMID: 19656489
7.  UV Irradiance and Albedo at Union Glacier Camp (Antarctica): A Case Study 
PLoS ONE  2014;9(3):e90705.
We report on the first spectral measurements of ultraviolet (UV) irradiance and the albedo at a Camp located in the southern Ellsworth Mountains on the broad expanse of Union Glacier (700 m altitude, 79° 46′ S; 82° 52′W); about 1,000 km from the South Pole. The measurements were carried out by using a double monochromator-based spectroradiometer during a campaign (in December 2012) meant to weight up the effect of the local albedo on the UV irradiance. We found that the albedo measured at noon was about 0.95 in the UV and the visible part of the spectrum. This high surface reflectivity led to enhancements in the UV index under cloudless conditions of about 50% in comparison with snow free surfaces. Spectral measurements carried out elsewhere as well as estimates retrieved from the Ozone Monitoring Instrument (OMI) were used for further comparisons.
doi:10.1371/journal.pone.0090705
PMCID: PMC3944898  PMID: 24598906
8.  A Novel Imaging System Permits Real-time in Vivo Tumor Bed Assessment After Resection of Naturally Occurring Sarcomas in Dogs 
Background
Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery.
Questions/purposes
To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin?
Methods
We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9–15 months).
Results
Fluorescence was apparent from all 10 tumors and ranged from 3 × 107 to 1 × 109 counts/millisecond/cm2. During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months.
Conclusions
These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections.
Clinical Relevance
The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control.
doi:10.1007/s11999-012-2560-8
PMCID: PMC3563778  PMID: 22972654
9.  Imaging primary mouse sarcomas after radiation therapy using cathepsin-activatable fluorescent imaging agents 
Purpose
Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS), and other tumors, undergo radiation therapy (RT) prior to surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue.
Methods and Materials
A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes and various tissues, including the tumor, were imaged using a handheld imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed.
Results
RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b positive tumor associated immune cells.
Conclusions
In this primary mouse model of STS, RT does not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes label tumor cells and tumor associated macrophages. Our results support including patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.
doi:10.1016/j.ijrobp.2012.12.007
PMCID: PMC3923491  PMID: 23391816
10.  Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell 
Cell Metabolism  2014;19(1):122-134.
Summary
Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.
Graphical Abstract
Highlights
•Silencing of miR-184 during insulin resistance promotes its target Ago2•Loss of Ago2 during insulin resistance blocks pancreatic β cell proliferation•Ago2 mediates the suppression of Cadm1 by miR-375 in the β cell•Administration of the ketogenic diet to ob/ob mice rescues miR-184 in islets
Tattikota et al. find that as β cells adapt to increased metabolic demand during insulin resistance in obesity, miR-184 is silenced to alleviate repression of its target Argonaute2, a component of the microRNA-induced silencing complex. Argonaute2 promotes compensatory β cell proliferation via miR-375 and its target genes, including the growth suppressor Cadm1.
doi:10.1016/j.cmet.2013.11.015
PMCID: PMC3945818  PMID: 24361012
11.  Detecting Force-Induced Molecular Transitions with Fluorescence Resonant Energy Transfer** 
Angewandte Chemie (International ed. in English)  2007;46(12):10.1002/anie.200604546.
doi:10.1002/anie.200604546
PMCID: PMC3870335  PMID: 17279589
biophysics; biosensors; FRET (fluorescence resonant energy transfer); optical tweezers; single-molecule studies
12.  Molecular origin of strain softening in cross-linked F-actin networks 
Two types of measurement are presented that relate molecular events to macroscopic behavior of F-actin networks. First, shear modulus is measured by oscillating an embedded microbead. Second, a microbead is translated at constant rate and transitions in the resisting force are observed. The loading rate dependence of the force at the transitions is similar to that of the molecular unbinding force, suggesting that they share a common origin. Reversibility tests of shear modulus provide further evidence that strain softening of F-actin networks is caused by force-induced rupture of cross-links.
PMCID: PMC3870339  PMID: 20866660
13.  Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes 
Cell metabolism  2012;16(4):435-448.
SUMMARY
A significant portion of the genome is transcribed as long non-coding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β-cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated, and show that they are an integral component of the β-cell differentiation and maturation program. We sequenced the mouse islet transcriptome, and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β-cell specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β-cell programming and diabetes pathophysiology.
doi:10.1016/j.cmet.2012.08.010
PMCID: PMC3475176  PMID: 23040067
14.  The miRNA Profile of Human Pancreatic Islets and Beta-Cells and Relationship to Type 2 Diabetes Pathogenesis 
PLoS ONE  2013;8(1):e55272.
Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis.
doi:10.1371/journal.pone.0055272
PMCID: PMC3555946  PMID: 23372846
15.  GATA believe it: new essential regulators of pancreas development 
The Journal of Clinical Investigation  2012;122(10):3469-3471.
Understanding the transcriptional mechanisms that underlie pancreas formation is central to the efforts to develop novel regenerative therapies for type 1 diabetes. Recently, mutations in the transcription factor GATA6 were unexpectedly shown to be the most common cause of human pancreas agenesis. In this issue of the JCI, Carrasco et al. and Xuan et al. investigate the role of Gata6 and its paralogue Gata4 in mouse embryonic pancreas and show that GATA factors are essential regulators of the proliferation, morphogenesis, and differentiation of multipotent pancreatic progenitors.
doi:10.1172/JCI65751
PMCID: PMC3461932  PMID: 23006323
16.  Plasticity of Adult Human Pancreatic Duct Cells by Neurogenin3-Mediated Reprogramming 
PLoS ONE  2012;7(5):e37055.
Aims/Hypothesis
Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.
Methods
The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.
Results
Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.
Conclusions/Interpretation
The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.
doi:10.1371/journal.pone.0037055
PMCID: PMC3351393  PMID: 22606327
17.  Passive and active microrheology for cross-linked F-actin networks in vitro 
Acta biomaterialia  2009;6(4):1207-1218.
Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity is not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and TIRF microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size, and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.
doi:10.1016/j.actbio.2009.10.044
PMCID: PMC2830389  PMID: 19883801
F-actin network; shear modulus; α-actinin; filamin; gelsolin
18.  EuroDia: a beta-cell gene expression resource 
Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms.
Database URL: http://eurodia.vital-it.ch
doi:10.1093/database/baq024
PMCID: PMC2963318  PMID: 20940178
19.  A map of open chromatin in human pancreatic islets 
Nature genetics  2010;42(3):255-259.
Tissue-specific transcriptional regulation is central to human disease1. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)2–4 coupled with high-throughput sequencing. We identified ~80,000 open chromatin sites. Comparison of islet FAIRE-seq to five non-islet cell lines revealed ~3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes exhibiting islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes (T2D)5, is located in islet-selective open chromatin. We show that rs7903146 heterozygotes exhibit allelic imbalance in islet FAIRE signal, and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements, and show that FAIRE-seq can guide identification of regulatory variants important for disease.
doi:10.1038/ng.530
PMCID: PMC2828505  PMID: 20118932
20.  Epistasis of Transcriptomes Reveals Synergism between Transcriptional Activators Hnf1α and Hnf4α 
PLoS Genetics  2010;6(5):e1000970.
The transcription of individual genes is determined by combinatorial interactions between DNA–binding transcription factors. The current challenge is to understand how such combinatorial interactions regulate broad genetic programs that underlie cellular functions and disease. The transcription factors Hnf1α and Hnf4α control pancreatic islet β-cell function and growth, and mutations in their genes cause closely related forms of diabetes. We have now exploited genetic epistasis to examine how Hnf1α and Hnf4α functionally interact in pancreatic islets. Expression profiling in islets from either Hnf1a+/− or pancreas-specific Hnf4a mutant mice showed that the two transcription factors regulate a strikingly similar set of genes. We integrated expression and genomic binding studies and show that the shared transcriptional phenotype of these two mutant models is linked to common direct targets, rather than to known effects of Hnf1α on Hnf4a gene transcription. Epistasis analysis with transcriptomes of single- and double-mutant islets revealed that Hnf1α and Hnf4α regulate common targets synergistically. Hnf1α binding in Hnf4a-deficient islets was decreased in selected targets, but remained unaltered in others, thus suggesting that the mechanisms for synergistic regulation are gene-specific. These findings provide an in vivo strategy to study combinatorial gene regulation and reveal how Hnf1α and Hnf4α control a common islet-cell regulatory program that is defective in human monogenic diabetes.
Author Summary
The transcriptional activity of each gene is typically determined by multiple transcription factors. This concept has been well established in studies of single genes. However, transcription factors do not simply regulate single genes, they also control broad gene programs that underlie cellular function and disease. Understanding how combinations of transcription factors interact at the level of cellular regulatory programs remains a challenge. Humans with mutations in the genes encoding for the transcription factors Hnf1α and Hnf4α develop similar forms of diabetes that result from abnormal insulin secretion, suggesting that the two factors might have related functions in insulin-producing islet-cells. We now show that Hnf1α or Hnf4α bind to a common set of genes and that islet-cells from mice in which either Hnf1α or Hnf4α has been selectively disrupted show abnormal expression of similar genes. By comparing the gene expression defects of mice with mutations in either Hnf1a, Hnf4a, or both genes, we determined that Hnf1α and Hnf4α regulate common target genes through synergistic mechanisms. These results thus provide insight into a regulatory network that fails in human diabetes. Similar genetic strategies can also be employed to unravel how other transcription factors interact functionally in native cellular contexts.
doi:10.1371/journal.pgen.1000970
PMCID: PMC2877749  PMID: 20523905
21.  LA BIOÉTICA COMO QUEHACER FILOSÓFICO 
Acta bioethica  2009;15(1):35-41.
El artículo examina el estatuto epistemológico de la bioética como disciplina académica. El autor sostiene que el estatuto epistemológico de un discurso lo determina la pregunta fundamental que se plantea y la respuesta que se busca, focos integradores del discurso. En el caso de la bioética, la pregunta fundamental es de índole moral. La bioética es pues una disciplina ética que tiene su hogar epistemológico en la filosofía. El autor también defiende el concepto de “éticas aplicadas”. Sugiere finalmente que el método de la bioética, sobre todo la que se hace desde nuestras latitudes, debería adoptar el círculo hermenéutico como metodología para su filosofar.
doi:10.4067/S1726-569X2009000100004
PMCID: PMC2867611  PMID: 20463860
bioética; ética aplicada; epistemología; estatuto epistemológico; transdisciplinariedad; círculo hermenéutico; bioethics; applied ethics; epistemology; epistemological statute; hermeneutic circle
22.  Functional Targets of the Monogenic Diabetes Transcription Factors HNF-1α and HNF-4α Are Highly Conserved Between Mice and Humans 
Diabetes  2009;58(5):1245-1253.
OBJECTIVE
The evolutionary conservation of transcriptional mechanisms has been widely exploited to understand human biology and disease. Recent findings, however, unexpectedly showed that the transcriptional regulators hepatocyte nuclear factor (HNF)-1α and -4α rarely bind to the same genes in mice and humans, leading to the proposal that tissue-specific transcriptional regulation has undergone extensive divergence in the two species. Such observations have major implications for the use of mouse models to understand HNF-1α– and HNF-4α–deficient diabetes. However, the significance of studies that assess binding without considering regulatory function is poorly understood.
RESEARCH DESIGN AND METHODS
We compared previously reported mouse and human HNF-1α and HNF-4α binding studies with independent binding experiments. We also integrated binding studies with mouse and human loss-of-function gene expression datasets.
RESULTS
First, we confirmed the existence of species-specific HNF-1α and -4α binding, yet observed incomplete detection of binding in the different datasets, causing an underestimation of binding conservation. Second, only a minor fraction of HNF-1α– and HNF-4α–bound genes were downregulated in the absence of these regulators. This subset of functional targets did not show evidence for evolutionary divergence of binding or binding sequence motifs. Finally, we observed differences between conserved and species-specific binding properties. For example, conserved binding was more frequently located near transcriptional start sites and was more likely to involve multiple binding events in the same gene.
CONCLUSIONS
Despite evolutionary changes in binding, essential direct transcriptional functions of HNF-1α and -4α are largely conserved between mice and humans.
doi:10.2337/db08-0812
PMCID: PMC2671044  PMID: 19188435
23.  Family and Population-Based Studies of Variation within the Ghrelin Receptor Locus in Relation to Measures of Obesity 
PLoS ONE  2010;5(4):e10084.
Background
The growth hormone secretagogue receptor (GHSR) is mediating hunger sensation when stimulated by its natural ligand ghrelin. In the present study, we tested the hypothesis that common and rare variation in the GHSR locus are related to increased prevalence of obesity and overweight among Whites.
Methodology/Principal Findings
In a population-based study sample of 15,854 unrelated, middle-aged Danes, seven variants were genotyped to capture common variation in an 11 kbp region including GHSR. These were investigated for their individual and haplotypic association with obesity. None of these analyses revealed consistent association with measures of obesity. A -151C/T promoter mutation in the GHSR was found in two unrelated obese patients. One family presented with complete co-segregation, but the other with incomplete co-segregation. The mutation resulted in an increased transcriptional activity (p<0.02) and introduction of a specific binding for Sp-1-like nuclear extracts relative to the wild type. The -151C/T mutation was genotyped in the 15,854 Danes with a minor allele frequency of 0.01%. No association with obesity in carriers (mean BMI: 27±4 kg/m2) versus non-carriers (mean BMI: 28±5 kg/m2) (p>0.05) could be shown.
Conclusions/Significance
In a population-based study sample of 15,854 Danes no association between GHSR genotypes and measures of obesity and overweight was found. Also, analyses of GHSR haplotypes lack consistent associations with obesity related traits. A rare functional GHSR promoter mutation variant was identified, yet there was no consistent relationship with obesity in neither family- nor population-based studies.
doi:10.1371/journal.pone.0010084
PMCID: PMC2852411  PMID: 20404923
24.  Hnf1α (MODY3) Controls Tissue-Specific Transcriptional Programs and Exerts Opposed Effects on Cell Growth in Pancreatic Islets and Liver▿ †  
Molecular and Cellular Biology  2009;29(11):2945-2959.
Heterozygous HNF1A mutations cause pancreatic-islet β-cell dysfunction and monogenic diabetes (MODY3). Hnf1α is known to regulate numerous hepatic genes, yet knowledge of its function in pancreatic islets is more limited. We now show that Hnf1a deficiency in mice leads to highly tissue-specific changes in the expression of genes involved in key functions of both islets and liver. To gain insights into the mechanisms of tissue-specific Hnf1α regulation, we integrated expression studies of Hnf1a-deficient mice with identification of direct Hnf1α targets. We demonstrate that Hnf1α can bind in a tissue-selective manner to genes that are expressed only in liver or islets. We also show that Hnf1α is essential only for the transcription of a minor fraction of its direct-target genes. Even among genes that were expressed in both liver and islets, the subset of targets showing functional dependence on Hnf1α was highly tissue specific. This was partly explained by the compensatory occupancy by the paralog Hnf1β at selected genes in Hnf1a-deficient liver. In keeping with these findings, the biological consequences of Hnf1a deficiency were markedly different in islets and liver. Notably, Hnf1a deficiency led to impaired large-T-antigen-induced growth and oncogenesis in β cells yet enhanced proliferation in hepatocytes. Collectively, these findings show that Hnf1α governs broad, highly tissue-specific genetic programs in pancreatic islets and liver and reveal key consequences of Hnf1a deficiency relevant to the pathophysiology of monogenic diabetes.
doi:10.1128/MCB.01389-08
PMCID: PMC2682018  PMID: 19289501
25.  The complete genomic sequence of an in vivo low replicating BLV strain 
Virology Journal  2009;6:120.
DNA was extracted from lamb lymphocytes that were infected in vivo with a BLV strain after inoculation with the peripheral blood mononuclear cells from a persistently sero-indeterminate, low viral load, BLV-infected Holstein cow (No. 41) from Argentina. The DNA was PCR amplified with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 41 DNA was cloned, sequenced, and compared phylogenetically to other BLV sequences including an in vivo high replicating strain (BLV ARG 38) from the same herd in Argentina. Characterization of BLV ARG 41's deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.
doi:10.1186/1743-422X-6-120
PMCID: PMC3224937  PMID: 19650931

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