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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a−/− cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a−/− cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

Author Summary

All cells in an organism share a common genome, yet distinct subsets of genes are transcribed in different cells. Selectivity of gene transcription is largely determined by transcription factors that bind to target genes and promote local changes in chromatin. Such changes are thought to be instrumental for transcription. Emerging evidence indicates that the position of genes in the 3-dimensional structure of the nucleus may also be important in transcriptional regulation. However, the role of transcription factors in gene positioning, and its possible relationship with chromatin modifications, is poorly understood. To examine this, we employed a genetic approach. We used mice lacking Hnf1α, a transcription factor gene that is mutated in an inherited form of diabetes. We studied genes that are directly bound by Hnf1α, as well as various control genomic regions, and determined their position in nuclear space in liver and insulin-producing β-cells. The results showed that the absence of Hnf1α causes local changes in the chromatin of target genes. At the same time, it modifies the position of target genes in nuclear space. The findings of this study lead us to propose a model whereby transcription factor dependent local chromatin modifications are linked to subnuclear gene positioning. They also revealed abnormal subnuclear positioning in a model of a human transcription factor disease.

Diabetes results from the absolute or relative deficiency of insulin-producing β cells. The prospect that non-β pancreatic cells could be harnessed to become β cells has led to interest in understanding the plasticity of pancreatic cells. Recent studies, however, have shown that adult β cells are largely derived from preexisting β cells. In this issue of the JCI, Desai et al. show that acinar cells, the major cell type in the pancreas, do not contribute to new β cells formed during pancreatic regeneration (see the related article beginning on page 971). These studies suggest that the fate of adult pancreatic cell lineages is immutable. However, also in this issue of the JCI, Collombat et al. demonstrate that inducing a single transcription factor named Arx in adult β cells causes these cells to undergo massive transdifferentiation into α and pancreatic polypeptide endocrine cells (see the related article beginning on page 961). This finding points to an unexpected plasticity of postnatal pancreatic endocrine cells.

Background

Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4α) and HNF1A/TCF1 (encoding HNF-1α), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.

Methods and Findings

We examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001); 56% (30/54) of HNF4A-mutation carriers were macrosomic compared with 13% (7/54) of non-mutation family members (p < 0.001). Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003). There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic β-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth.

Conclusions

HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.

HNF4A mutations were found to be associated with a considerable increase in birthweight and macrosomia, and were a cause of neonatal hypoglycaemia.

Editors' Summary

Background.

MODY, or maturity-onset diabetes of the young, is a particular subtype of diabetes; only a few percent of people with diabetes are thought to have this subtype. The condition comes about as a result of a mutation in one of six genes. Generally, people with MODY have high glucose (sugar) levels in the blood, and the typical symptoms of diabetes, such as increased thirst and urination, typically develop when the person is below the age of 25 y. Two of the genes that are known to cause MODY are mutant forms of HNF4A and HNF1A. The proteins that are encoded by these two genes control insulin levels produced by the pancreas; when these genes are mutated, not enough insulin is produced. Without enough insulin to control blood sugar, levels rise, leading to the symptoms of diabetes. However, MODY can be managed by many of the same interventions as other types of diabetes, such as diet, exercise, drug treatments, and insulin injections.

Why Was This Study Done?

Although the evidence shows that individuals who carry mutations in HNF4A and HNF1A do not produce enough insulin and therefore have higher glucose levels in their blood, there were some tantalizing suggestions from mouse experiments that this might not be the whole story. Specifically, the researchers suspected that during embryonic development, mutations in HNF4A or HNF1A might actually cause higher insulin levels. Too much insulin during development of a fetus is known to cause it to gain weight, resulting in a baby that is larger than the average size for its age. Larger babies are risky for both the baby and the mother. The researchers doing this study wanted to understand more precisely what the links were between the forms of MODY caused by HNF4A and HNF1A mutations, and birth-weight and blood-sugar levels.

What Did the Researchers Do and Find?

In this study, the researchers examined 15 families in which some family members had MODY caused by a mutation in HNF4A. They compared the birthweight for family members carrying the mutation (54 people) against the birthweight for those who did not (54 people). A similar comparison was done for 38 families in which some members had a different form of MODY, this time caused by a mutation in HNF1A. The results showed that the birthweight of family members who carried a mutation in HNF4A was, on average, 790 g higher than the birthweight of family members who didn't carry the mutation. Low blood-sugar levels at birth were also more common in people carrying the HNF4A mutation as compared to people who did not. However, the HNF1A mutation did not seem to be associated with greater birthweight or low blood-sugar levels at birth. Finally, in order to understand these findings further, the researchers created embryonic mice carrying mutations in the mouse equivalent of HNF4A. These embryos produced more insulin than normal mouse embryos and, after birth, were more likely to have low blood-sugar levels.

What Do These Findings Mean?

These findings show that there is a link between mutations in HNF4A, but not in HNF1A, and increased birthweight. The increase found in this study is quite substantial (a median weight of 4,660 g in the affected babies; a birthweight of more than 4,000 g is generally considered large). The results suggest that in human embryos with a mutated form of HNF4A, too much insulin is produced during development, causing faster growth and a higher chance of the baby being born with low blood-sugar levels. This is an unexpected finding, because later in life the HNF4A mutation causes lower insulin levels. Therefore, the biochemical pathways causing this type of MODY seem to be quite complicated, and further research will need to be done to fully understand them. Crucially, the research also suggests that pregnant women carrying HNF4A mutations should be closely followed to check their baby's growth and minimize the chance of complications. Doctors and families should also consider doing a genetic test for HNF4A if a baby has low blood-sugar levels and if there is a family history of diabetes; this would increase the chance of diagnosing MODY early.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed 0040118.

In a related Perspective in PLoS Medicine, Benjamin Glaser discusses causes of type 2 diabetes mellitus in the context of this study's findings

The US National Institute of Diabetes and Digestive and Kidney Diseases has pages of information on different types of diabetes

Wikipedia has an entry on Maturity Onset Diabetes of the Young (MODY) (note that Wikipedia is an internet encyclopedia that anyone can edit)

Diabetes Research Department, Peninsula Medical School, Exeter, UK provides information for patients and doctors on genetic types of diabetes; the website is maintained by the research group carrying out this study

Information from the Centers for Disease Control and Prevention on diabetes and pregnancy

Mutations in the genes encoding hepatocyte nuclear factor 4α (HNF-4α) and HNF-1α impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4α is known to be an essential positive regulator of HNF-1α. More recent data demonstrates that HNF-4α expression is dependent on HNF-1α in mouse pancreatic islets and exocrine cells. This effect is mediated by binding of HNF-1α to a tissue-specific promoter (P2) located 45.6 kb upstream from the previously characterized Hnf4α promoter (P1). Here we report that the expression of HNF-4α in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G → A mutation in a conserved nucleotide position of the HNF-1α binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1α, and consequently in reduced HNF-1α–dependent activation. These findings provide genetic evidence that HNF-1α serves as an upstream regulator of HNF-4α and interacts directly with the P2 promoter in human pancreatic cells. Furthermore, they indicate that this regulation is essential to maintain normal pancreatic function.

Mutations in the gene encoding hepatic nuclear factor 1-α (HNF1-α) cause a subtype of human diabetes resulting from selective pancreatic β-cell dysfunction. We have analyzed mice lacking HNF1-α to study how this protein controls β-cell-specific transcription in vivo. We show that HNF1-α is essential for the expression of glut2 glucose transporter and L-type pyruvate kinase (pklr) genes in pancreatic insulin-producing cells, whereas in liver, kidney, or duodenum tissue, glut2 and pklr expression is maintained in the absence of HNF1-α. HNF1-α nevertheless occupies the endogenous glut2 and pklr promoters in both pancreatic islet and liver cells. However, it is indispensable for hyperacetylation of histones in glut2 and pklr promoter nucleosomes in pancreatic islets but not in liver cells, where glut2 and pklr chromatin remains hyperacetylated in the absence of HNF1-α. In contrast, the phenylalanine hydroxylase promoter requires HNF1-α for transcriptional activity and localized histone hyperacetylation only in liver tissue. Thus, different HNF1-α target genes have distinct requirements for HNF1-α in either pancreatic β-cells or liver cells. The results indicate that HNF1-α occupies target gene promoters in diverse tissues but plays an obligate role in transcriptional activation only in cellular- and promoter-specific contexts in which it is required to recruit histone acetylase activity. These findings provide genetic evidence based on a live mammalian system to establish that a single activator can be essential to direct nucleosomal hyperacetylation to transcriptional targets.

Abstract

BACKGROUND: The goal of limb-sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization. METHODS: A primary mouse model of soft tissue sarcoma and a wide field-of-view imaging device were used to investigate a series of exogenously administered, near-infrared (NIR) fluorescent probes activated by cathepsin proteases for real-time intraoperative imaging. RESULTS: The authors demonstrated that exogenously administered cathepsin-activated probes can be used for image-guided surgery to identify microscopic residual NIR fluorescence in the tumor beds of mice. The presence of residual NIR fluorescence was correlated with microscopic residual sarcoma and local recurrence. The removal of residual NIR fluorescence improved local control. CONCLUSIONS: The authors concluded that their technique has the potential to be used for intraoperative image-guided surgery to identify microscopic residual disease in patients with cancer. Cancer 2012. © 2012 American Cancer Society.