Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.
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Islet1; Heart development; Cardiac progenitor cell; Cardiac stem cell
The generation of mature cell types during pancreatic development depends on the expression of many regulatory and signaling proteins. In this study, we tested the hypothesis that the transcriptional regulator Islet-1 (Isl-1), whose expression is first detected in the mesenchyme and epithelium of the developing pancreas and is later restricted to mature islet cells, is involved in the terminal differentiation of islet cells and maintenance of islet mass.
RESEARCH DESIGN AND METHODS
To investigate the role of Isl-1 in the pancreatic epithelium during the secondary transition, Isl-1 was conditionally and specifically deleted from embryonic day 13.5 onward using Cre/LoxP technology.
Isl-1–deficient endocrine precursors failed to mature into functional islet cells. The postnatal expansion of endocrine cell mass was impaired, and consequently Isl-1 deficient mice were diabetic. In addition, MafA, a potent regulator of the Insulin gene and β-cell function, was identified as a direct transcriptional target of Isl-1.
These results demonstrate the requirement for Isl-1 in the maturation, proliferation, and survival of the second wave of hormone-producing islet cells.
The anterior heart field (AHF), which contributes to the outflow tract and right ventricle of the heart, is defined in part by expression of the LIM homeobox transcription factor Isl-1. The importance of Isl-1–positive cells in cardiac development and homeostasis is underscored by the finding that these cells are required for cardiac development and act as cardiac stem/progenitor cells within the postnatal heart. However, the molecular pathways regulating these cells’ expansion and differentiation are poorly understood. We show that Isl-1–positive AHF progenitor cells in mice were responsive to Wnt/β-catenin signaling, and these responsive cells contributed to the outflow tract and right ventricle of the heart. Loss of Wnt/β-catenin signaling in the AHF caused defective outflow tract and right ventricular development with a decrease in Isl-1–positive progenitors and loss of FGF signaling. Conversely, Wnt gain of function in these cells led to expansion of Isl-1–positive progenitors with a concomitant increase in FGF signaling through activation of a specific set of FGF ligands including FGF3, FGF10, FGF16, and FGF20. These data reveal what we believe to be a novel Wnt-FGF signaling axis required for expansion of Isl-1–positive AHF progenitors and suggest future therapies to increase the number and function of these cells for cardiac regeneration.
Islet1 (Isl1) is a LIM homedomain protein that plays a pivotal role in cardiac progenitors of the second heart field. Here, lineage studies with an inducible isl1-cre demonstrated that most Isl1 progenitors have migrated into the heart by E9. Although Isl1 expression is downregulated in most cardiac progenitors as they differentiate, analysis of an isl1-nlacZ mouse and coimmunostaining for Isl1 and lineage markers demonstrated that Isl1 is expressed in distinct subdomains of the heart, and in diverse cardiovascular lineages. Isl1 expression was observed in myocardial lineages of the distal outflow tract, atrial septum, and in sinoatrial and atrioventricular node. The myocardialized septum of the outflow tract was found to derive from Isl1 expressing cells. Isl1 expressing cells also contribute to endothelial and vascular smooth muscle lineages including smooth muscle of the coronary vessels. Our data indicate that Isl1 is a specific marker for a subset of pacemaker cells at developmental stages examined, and suggest genetic heterogeneity within the central conduction system and coronary smooth muscle. Our studies suggest a role for Isl1 in these distinct domains of expression within the heart.
Cardiac progenitor; Lineage; Isl1; pacemaker; Coronary smooth muscle; Tamoxifen; Inducible Cre
Cardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency.
We sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1+/Nkx2.5+ CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1+/Nkx2.5+ CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1+/Flt4+ best identified and facilitated enrichment for Isl1+/Nkx2.5+ CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1+/Flt4+ CPCs differentiated into all three cardiovascular lineages in vitro. Flt1+/Flt4+ CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs).
The cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1+/Nkx2.5+ CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts.
Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.
The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs1–3, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation4, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.
β-Catenin; Notch1; Isl1; cardiac progenitors; Myocd
The cells of the second heart field (SHF) contribute to the outflow tract and right ventricle, as well as to parts of the left ventricle and atria. Isl1, a member of the LIM-homeodomain transcription factor family, is expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development. Isl1 is required for the survival and migration of SHF-derived cells into the early developing heart at the inflow and outflow poles. Despite this important role for Isl1 in early heart formation, the transcriptional regulation of Isl1 has remained largely undefined. Therefore, to identify transcription factors that regulate Isl1 expression in vivo, we screened the conserved noncoding sequences from the mouse Isl1 locus for enhancer activity in transgenic mouse embryos. Here, we report the identification of an enhancer from the mouse Isl1 gene that is sufficient to direct expression to the SHF and its derivatives. The Isl1 SHF enhancer contains three consensus Forkhead transcription factor binding sites that are efficiently and specifically bound by Forkhead transcription factors. Importantly, the activity of the enhancer is dependent on these three Forkhead binding sites in transgenic mouse embryos. Thus, these studies demonstrate that Isl1 is a direct transcriptional target of Forkhead transcription factors in the SHF and establish a transcriptional pathway upstream of Isl1 in the SHF.
The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.
Motor neuron; V2 interneuron; Cranial ganglia; Cell death; Isl1; Axon growth
Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotrophs to stimulate synthesis and secretion of the luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used (nuclear factor of activated T-cells 2 (NFAT2)-emerald fluorescent protein) to monitor GnRH signaling. Increasing [Ca2+]i causes calmodulin/calcineurin-dependent nuclear NFAT translocation, a response involving proteins (calmodulins and NFATs) that decode frequency in other systems. Using live cell imaging, pulsatile GnRH caused dose- and frequency-dependent increases in nuclear NFAT2-emerald fluorescent protein, and at low frequency, translocation simply tracked GnRH exposure (albeit with slower kinetics). At high frequency (30-min intervals), failure to return to basal conditions before repeat stimulation caused integrative tracking, illustrating how the relative dynamics of up- and downstream signals can increase efficiency of GnRH action. Mathematical modeling predicted desensitization of GnRH effects on [Ca2+]i and that desensitization would increase with dose, frequency, and receptor number, but no such desensitization was seen in HeLa and/or LβT2 cells possibly because pulsatile GnRH did not reduce receptor expression (measured by immunofluorescence). GnRH also caused dose- and frequency-dependent activation of αGSU, luteinizing hormone β, and follicle-stimulating hormone β luciferase reporters, effects that were blocked by calcineurin inhibition. Pulsatile GnRH also activated an NFAT-responsive luciferase reporter, but this response was directly related to cumulative pulse duration. This together with the lack of desensitization of translocation responses suggests that NFAT may mediate GnRH action but is not a genuine decoder of GnRH pulse frequency.
Islet 1 (ISL1), a LIM-homeodomain transcription factor is essential for promoting pancreatic islets proliferation and maintaining endocrine cells survival in embryonic and postnatal pancreatic islets. However, how ISL1 exerts the role in adult islets is, to date, not clear.
Our results show that ISL1 expression was up-regulated at the mRNA level both in cultured pancreatic cells undergoing glucose oxidase stimulation as well in type 1 and type 2 diabetes mouse models. The knockdown of ISL1 expression increased the apoptosis level of HIT-T15 pancreatic islet cells. Using HIT-T15 and primary adult islet cells as cell models, we show that ISL1 promoted adult pancreatic islet cell proliferation with increased c-Myc and CyclinD1 transcription, while knockdown of ISL1 increased the proportion of cells in G1 phase and decreased the proportion of cells in G2/M and S phases. Further investigation shows that ISL1 activated both c-Myc and CyclinD1 transcription through direct binding on their promoters.
ISL1 promoted adult pancreatic islet cell proliferation and probably by activating c-Myc and CyclinD1 transcription through direct binding on their promoters. Our findings extend the knowledge about the crucial role of ISL1 in maintaining mature islet cells homeostasis. Our results also provide insights into the new regulation relationships between ISL1 and other growth factors.
Isl1 is a LIM/homeodomain transcription factor with critical roles for the development of the heart, the nervous system and the pancreas. Both deficiency and mis-expression of Isl1 cause profound developmental defects, demonstrating the importance of proper regulation of Isl1 gene expression during development. In order to understand the mechanisms that control Isl1 expression during embryogenesis and in tissue differentiation, we initiated a screen for gene regulatory elements in the Isl1 locus using a novel dual reporter gene vector that allows screens of large genomic regions through reporter gene assays in vitro and in vivo. We identified regions from the Isl1 gene locus that confer transcriptional activity in pancreatic cell lines in vitro. Using transgenic mice, we furthermore discovered an enhancer with in vivo specificity for the developing heart, as well as visceral and posterior mesoderm. Our findings further suggest that Foxo1 as well as Gata4 contribute to the activity of this enhancer in the developing embryo. We conclude that Isl1 gene expression is controlled in modular fashion by several elements with distinct functionality. Embryonic Isl1 expression in several tissues of mesodermal origin is driven by a specific enhancer that is located 3-6kb downstream of the gene.
transgenic reporter; enhancer; heart mesoderm; cardiac crescent; lateral mesoderm
Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON- and OFF-bipolar (>76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of “wild-type” cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON- and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIM-homeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.
retina; retinal bipolar cell; transcription factor; differentiation; ERG (electroretinogram); optomotor behavior; amacrine; retinal ganglion cell
Congenital heart disease (CHD) is the most common birth abnormality and the etiology is unknown in the overwhelming majority of cases. ISLET1 (ISL1) is a transcription factor that marks cardiac progenitor cells and generates diverse multipotent cardiovascular cell lineages. The fundamental role of ISL1 in cardiac morphogenesis makes this an exceptional candidate gene to consider as a cause of complex congenital heart disease. We evaluated whether genetic variation in ISL1 fits the common variant–common disease hypothesis. A 2-stage case-control study examined 27 polymorphisms mapping to the ISL1 locus in 300 patients with complex congenital heart disease and 2,201 healthy pediatric controls. Eight genic and flanking ISL1 SNPs were significantly associated with complex congenital heart disease. A replication study analyzed these candidate SNPs in 1,044 new cases and 3,934 independent controls and confirmed that genetic variation in ISL1 is associated with risk of non-syndromic congenital heart disease. Our results demonstrate that two different ISL1 haplotypes contribute to risk of CHD in white and black/African American populations.
The mammalian retina is comprised of six major neuronal cell types and is subdivided into more morphological and physiological subtypes. The transcriptional machinery underlying these subtype fate choices is largely unknown. The LIM-homeodomain protein, Isl1, plays an essential role in central nervous system (CNS) differentiation but its relationship to retinal neurogenesis remains unknown. We report here its dynamic spatiotemporal expression in the mouse retina. Among bipolar interneurons, Isl1 expression commences at postnatal day (P)5 and is later restricted to ON-bipolar cells. The intensity of Isl1 expression is found to segregate the pool of ON-bipolar cells into rod and ON-cone bipolar cells with higher expression in rod bipolar cells. As bipolar cell development proceeds from P5–10 the colocalization of Isl1 and the pan-bipolar cell marker Chx10 reveals the organization of ON-center bipolar cell nuclei to the upper portion of the inner nuclear layer. Further, whereas Isl1 is predominantly a ganglion cell marker prior to embryonic day (E)15.5, at E15.5 and later its expression in nonganglion cells expands. We demonstrate that these Isl1-positive, nonganglion cells acquire the expression of amacrine cell markers embryonically, likely representing nascent cholinergic amacrine cells. Taken together, Isl1 is expressed during the maturation of and is later maintained in retinal ganglion cells and subtypes of amacrine and bipolar cells where it may function in the maintenance of these cells into adulthood. J. Comp. Neurol. 503: 182–197, 2007.
ON-bipolar cells; Chx10; amacrine cell; retina; neurogenesis; transcription factors; subtype markers
Multipotent Isl1+ heart progenitors give rise to three major cardiovascular cell types; cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that an Isl1+/SLN+ transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1+/SLN+ cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin.
cardiogenesis; myogenic progenitor; smooth muscle; great vessel; plasticity
We examine results of gain-of-function experiments on retinocollicular maps in knock-in mice [Brown et al. (2000) Cell 102:77]. In wild-type mice the temporal-nasal axis of retina is mapped to the rostral-caudal axis of superior colliculus. The established map is single-valued, which implies that each point in retina maps to a unique termination zone in superior colliculus. In homozygous Isl2/EphA3 knock-in mice the map is double-valued, which means that each point on retina maps to two termination zones in superior colliculus. This is because about 50 percent of cells in retina express Isl2, and two types of projections, wild-type and Isl2/EphA3 positive, form two branches of the map. In heterozygous Isl2/EphA3 knock-ins the map is intermediate between the homozygous and wild-type: it is single-valued in temporal and double-valued in the nasal parts of retina. In this study we address possible reasons for such a bifurcation of the map.
We study the map formation using stochastic model based on Markov chains. In our model the map undergoes a series of reconstructions with probabilities dependent upon a set of chemical cues. Our model suggests that the map in heterozygotes is single-valued in temporal region of retina for two reasons. First, the inhomogeneous gradient of endogenous receptor in retina makes the impact of exogenous receptor less significant in temporal retina. Second, the gradient of ephrin in the corresponding region of superior colliculus is smaller, which reduces the chemical signal-to-noise ratio. We predict that if gradient of ephrin is reduced by a genetic manipulation, the single-valued region of the map should extend to a larger portion of temporal retina, i.e. the point of transition between single-and doulble-valued maps should move to a more nasal position in Isl2-EphA3 heterozygotes.
We present a theoretical model for retinocollicular map development, which can account for intriguing behaviors observed in gain-of-function experiments by Brown et al., including bifurcation in heterozygous Isl2/EphA3 knock-ins. The model is based on known chemical labels, axonal repulsion/competition, and stochasticity. Possible mapping in Isl2/EphB knock-ins is also discussed.
Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-phosphorylated Stat3 was colocalized with Isl1 in the nucleus of the mouse motor neurons in spinal cord after nerve injury. Correspondingly, electroporation of Isl1 and Stat3 into the neural tube of chick embryos resulted in the activation of a reporter gene expression controlled by a Stat3 regulatory sequence, and cotransfection of Isl1 and Stat3 promoted the proliferation of the mouse motor neuron cells. Our data suggest a novel role of Isl1 as an adaptor for Jak1 and Stat3 and reveal a possible functional link between LIM homeodomain transcription factors and the Jak-Stat pathway.
LIM-homeodomain (HD) and POU-HD transcription factors play critical roles in neurogenesis. However, it remains largely unknown how they cooperate in this process and what downstream target genes they regulate. Here we show that ISL1, a LIM-HD protein, is co-expressed with BRN3B, a POU-HD factor, in nascent, post-mitotic retinal ganglion cells (RGCs). Similar to the Brn3b-null retinas, retina-specific deletion of Isl1 results in the apoptosis of a majority of RGCs and in RGC axon guidance defects. The Isl1 and Brn3b double null mice display more severe retinal abnormalities with a near complete loss of RGCs, indicating the synergistic functions of these two factors. Furthermore, we show that both Isl1 and Brn3b function downstream of Math5 to regulate the expression of a common set of RGC-specific genes. Whole retina chromatin immunoprecipitation and in vitro transactivation assays reveal that ISL1 and BRN3B concurrently bind to and synergistically regulate the expression of a common set of RGC-specific genes. Thus, our results uncover a novel regulatory mechanism of BRN3B and ISL1 in RGC differentiation.
LIM-homeodomain; POU-domain; Math5; Atoh7; Pou4f2; RGC; retinal development; transcription factor
The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.
The U6 RNA internal stem-loop (U6 ISL) is a highly conserved domain of the spliceosome that is important for pre-mRNA splicing. The U6 ISL contains an internal loop that is in equilibrium between two conformations controlled by the protonation state of an adenine (pKa = 6.5). Lower pH favors formation of a protonated C-A+ wobble pair and base flipping of the adjacent uracil. Higher pH favors stacking of the uracil, and allows an essential metal ion to bind at this position. Here, we define the minimal energy path for this conformational transition. To do this, we solved the U6 ISL structure at higher pH (8.0), in order to eliminate interference from the low pH conformer. This structure reveals disruption of the protonated C-A+ pair and formation of a new C-U pair, which explains the preference for a stacked uracil at higher pH. Next, we used Nudged Elastic Band (NEB) molecular dynamics simulations to calculate the minimum energy path between the two conformations. Our results indicate that the C-U pair is dynamic, which allows formation of the more stable C-A+ pair upon adenine protonation. After formation of the C-A+ pair, the unpaired uracil follows a minor groove base flipping pathway. Molecular dynamics simulations suggest that the extrahelical uracil is stabilized by contacts with the adjacent helix.
U6 RNA; RNA Structure; NMR; Molecular Dynamics; Nudged Elastic Band; Base flipping
U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3′ intramolecular stem–loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson–Crick base pair (C67·G80), and disrupts a protonated C67·A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (ΔΔGfold = −3.6 ± 1.9 kcal/mol), consistent with formation of the Watson–Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem–loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.
An introduction and overview of the focus, goals and overall mission of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) is given.
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This issue of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.
SSGCID; structural genomics; structure-based drug design; infectious diseases; pathogens; emerging and re-emerging diseases
A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson’s disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.
α-Synuclein; phosphoprotein; glycoprotein; proteomics
Hydrogen peroxide (H2O2) can serve as a beneficial signaling agent or toxin depending on its concentration and location within a cell or organism. Methods to measure the localized accumulation of H2O2 in living specimens remain limited. Motivated to meet this need, we have developed a new type of nuclear-localized fluorescent probe for H2O2, Nuclear Peroxy Emerald 1 (NucPE1), to selectively interrogate ROS fluxes within this sensitive organelle. NucPE1 selectively accumulates in the nuclei of a variety of mammalian cell lines as well as in whole model organisms like C. clegans, where it can respond to subcellular changes in H2O2 fluxes. Moreover, in vivo NucPE1 imaging reveals a reduction in nuclear H2O2 levels in worms overexpressing sir-2.1 compared to wildtype congeners, supporting a link between this longevity-promoting sirtuin protein and enhanced regulation of nuclear ROS pools.