Brad G. Hoffman

Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression.

Maintenance of β-Cell Maturity and Plasticity in the Adult Pancreas: Developmental Biology Concepts in Adult Physiology

Diabetes occurs when the pancreas fails to produce and secrete sufficient insulin for the maintenance of glucose homeostasis. Although many factors required for β-cell development have been elucidated, we know surprisingly little regarding the mechanisms that maintain the differentiated state of adult β-cells. Despite years of fruitful research, many hurdles remain before we can replace failing β-cells in patients. Efforts to produce new β-cells will benefit from detailed knowledge of their differentiation, maturation, maintenance, heterogeneity, and plasticity. Studies of single cells and conditional knockout mice reveal surprising relationships between the different islet cell types and previously unappreciated roles for transcription factors and soluble factors in β-cell maintenance. Herein we highlight numerous innovative efforts made to identify the core mechanisms responsible for β-cell phenotypic maintenance and compare these with other long-lived cell types. In the adult pancreas, physical β-cell mass is determined by the balance of β-cell birth, differentiation, size, and death. The prenatal origins and development of β-cells (1,2), the molecular mechanisms of adult β-cell proliferation (3,4), and programmed β-cell death (5) have all been reviewed recently and will not be revisited here. The maintenance and adaptation of β-cell mass after birth involves the addition of new β-cells (6). Cells with robust staining for insulin have been shown to slowly proliferate in vivo and in vitro (7–9). It has been reported that β-cells have an extremely long life span under typical conditions and proliferation plays a diminishing role with advancing age (7,10,11). The apparent low rate of β-cell replication has led some groups to suggest that adult β-cells arise from a pool of non–β-cell progenitors (12). Because this topic has been hotly debated and reviewed recently (13), we will only briefly discuss studies relevant to other aspects of this review. …

Paracrine signalling loops in adult human and mouse pancreatic islets: netrins modulate beta cell apoptosis signalling via dependence receptors.

Aims/hypothesisAdult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets.MethodsGene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined.ResultsWe identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling.Conclusions/interpretationOur results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.

Genome-wide identification of DNA-protein interactions using chromatin immunoprecipitation coupled with flow cell sequencing

The transcriptional networks underlying mammalian cell development and function are largely unknown. The recently described use of flow cell sequencing devices in combination with chromatin immunoprecipitation (ChIP-seq) stands to revolutionize the identification of DNA-protein interactions. As such, ChIP-seq is rapidly becoming the method of choice for the genome-wide localization of histone modifications and transcription factor binding sites. As further studies are performed, the information generated by ChIP-seq is expected to allow the development of a framework for networks describing the transcriptional regulation of cellular development and function. However, to date, this technology has been applied only to a small number of cell types, and even fewer tissues, suggesting a huge potential for novel discovery in this field.

Identification of transcripts with enriched expression in the developing and adult pancreas.

BackgroundDespite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.ResultsWe used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.ConclusionTaken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

Antagonistic effects of Grg6 and Groucho/TLE on the transcription repression activity of brain factor 1/FoxG1 and cortical neuron differentiation.

ABSTRACT Groucho (Gro)/TLE transcriptional corepressors are involved in a variety of developmental mechanisms, including neuronal differentiation. They contain a conserved C-terminal WD40 repeat domain that mediates interactions with several DNA-binding proteins. In particular, Gro/TLE1 interacts with forkhead transcription factor brain factor 1 (BF-1; also termed FoxG1). BF-1 is an essential regulator of neuronal differentiation during cerebral cortex development and represses transcription together with Gro/TLE1. Gro/TLE-related gene product 6 (Grg6) shares with Gro/TLEs a conserved WD40 repeat domain but is more distantly related at its N-terminal half. We demonstrate that Grg6 is expressed in cortical neural progenitor cells and interacts with BF-1. In contrast to Gro/TLE1, however, Grg6 does not promote, but rather suppresses, BF-1-mediated transcriptional repression. Consistent with these observations, Grg6 interferes with the binding of Gro/TLE1 to BF-1 and does not repress transcription when targeted to DNA. Moreover, coexpression of Grg6 and BF-1 in cortical progenitor cells leads to a decrease in the number of proliferating cells and increased neuronal differentiation. Conversely, Grg6 knockdown by RNA interference causes decreased neurogenesis. These results identify a new role for Grg6 in cortical neuron development and establish a functional link between Grg6 and BF-1.

Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.

Identification and analysis of murine pancreatic islet enhancers

Aims/hypothesisThe paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression.MethodsWe combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets.ResultsWe identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs).Conclusions/interpretationsOur results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Probabilistic inference for nucleosome positioning with MNase-based or sonicated short-read data.

We describe a model-based method, PING, for predicting nucleosome positions in MNase-Seq and MNase- or sonicated-ChIP-Seq data. PING compares favorably to NPS and TemplateFilter in scalability, accuracy and robustness to low read density. To demonstrate that PING predictions from widely available sonicated data can have sufficient spatial resolution to be to be useful for biological inference, we use Illumina H3K4me1 ChIP-seq data to detect changes in nucleosome positioning around transcription factor binding sites due to tamoxifen stimulation, to discriminate functional and non-functional transcription factor binding sites more effectively than with enrichment profiles, and to confirm that the pioneer transcription factor Foxa2 associates with the accessible major groove of nucleosomal DNA.

Expression of Groucho/TLE proteins during pancreas development

BackgroundThe full-length mammalian homologs of groucho, Tle1, 2, 3, and 4, act as transcriptional corepressors and are recruited by transcription factors containing an eh1 or WRPW/Y domain. Many transcription factors critical to pancreas development contain a Gro/TLE interaction domain and several have been shown to require Gro/TLE interactions for proper function during neuronal development. However, a detailed analysis of the expression patterns of the Gro/TLE proteins in pancreas development has not been performed. Moreover, little is known about the ability of Gro/TLE proteins to interact with transcription factors in the pancreas.ResultsWe describe the expression of Gro/TLE family members, and of 34 different transcription factors that contain a Gro/TLE interaction motif, in the pancreas utilizing nine SAGE libraries created from the developing and adult pancreas, as well as the GenePaint database. Next, we show the dynamic expression of Tle1, 2, 3, 4, 5 and 6 during pancreas development by qRT-PCR. To further define the cell-type specificity of the expression of these proteins we use immunofluorescence to co-localize them with Pdx1 at embryonic day 12.5 (E12.5), Ngn3 at E14.5, Pdx1, Nkx2-2, Insulin, Glucagon, Pancreatic polypeptide and Somatostatin at E18.5, as well as Insulin and Glucagon in the adult. We then show that Tle2 can interact with Nkx2-2, Hes1, Arx, and Nkx6-1 which are all critical factors in pancreas development. Finally, we demonstrate that Tle2 modulates the repressive abilities of Arx in a β-cell line.ConclusionAlthough Tle1, 2, 3, and 4 show overlapping expression in pancreatic progenitors and in the adult islet, the expression of these factors is restricted to different cell types during endocrine cell maturation. Of note, Tle2 and Tle3 are co-expressed with Gro/TLE interaction domain containing transcription factors that are essential for endocrine pancreas development. We further demonstrate that Tle2 can interact with several of these factors and that Tle2 modulate Arxs repressive activity. Taken together our studies suggest that Gro/TLE proteins play a role in the repression of target genes during endocrine cell specification.