Full-length Isoform Research Articles

The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1G93A Models, and Its Inhibition Decreases the Expression of Proinflammatory Genes.

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.

Effects of neuregulin GGF2 (cimaglermin alfa) dose and treatment frequency on left ventricular function in rats following myocardial infarction

Neuregulins are important growth factors involved in cardiac development and response to stress. Certain isoforms and fragments of neuregulin have been found to be cardioprotective. The effects of a full-length neuregulin-1β isoform, glial growth factor 2 (GGF2; USAN/INN; also called cimaglermin) were investigated in vitro. Various dosing regimens were then evaluated for their effects on left ventricular (LV) function in rats with surgically-induced myocardial infarction. In vitro, GGF2 bound with high affinity to erythroblastic leukemia viral oncogene (ErbB) 4 receptors, potently promoted Akt phosphorylation, as well as reduced cell death following doxorubicin exposure in HL1 cells. Daily GGF2 treatment beginning 7–14 days after left anterior descending coronary artery ligation produced improvements in LV ejection fraction and other measures of LV function and morphology. The improvements in LV function (e.g. 10% point increase in absolute LV ejection fraction) with GGF2 were dose-dependent. LV performance was substantially improved when GGF2 treatment was delivered infrequently, despite a serum half-life of less than 2h and could be maintained for more than 10 months with treatment once weekly or once every 2 weeks. These studies confirm previous findings that GGF2 may improve contractile performance in the failing rat heart and that infrequent exposure to GGF2 may improve LV function and impact remodeling in the failing myocardium. GGF2 is now being developed for the treatment of heart failure in humans.

Identification of the binding domains and key amino acids for the interaction of the transcription factors BmPOUM2 and BmAbd-A in Bombyx mori

The transcription factor BmPOUM2 interacted with another transcription factor BmAbd-A to regulate the expression of the wing cuticle protein gene BmWCP4 in Bombyx mori. In this study, the binding domains and amino acids for the interaction between BmPOUM2 and BmAbd-A were reported. Two isoforms of BmPOUM2 were identified. The short isoform (BmPOUM2-S) lacks a 114-amino acid sequence containing a POU-homeodomain and a nuclear localization signal peptide (NLS), as compared to the full-length isoform (BmPOUM2). Both BmPOUM2 and BmPOUM2-S proteins bound to the BmAbd-A through the POU-specific domain. When the six amino acids (Lys166, Gly173, Gln176, Ser192, Glu200 and Asn208) that are highly conserved in POU family genes were mutated, BmPOUM2 did not bind to BmAbd-A. BmAbd-A interacted with BmPOUM2 by the homeobox domain or the LCR2 (low complexity region) domain. When seven amino acids (Phe156/248, His158/250, Ala175/263, Cys180/265, Glu190/268, Trp196/274 and Val214/289) that are shared in the homeobox and LCR2 domains were mutated, BmAbd-A did not bind to BmPOUM2. Overexpression of either BmPOUM2 or BmAbd-A or both increased the activity of BmWCP4 promoter in CHO cells. ChIP assay and EMSA showed that BmAbd-A protein bound to the Hox cis-regulatory element in the BmWCP4 promoter, while the BmPOUM2 bound to the nearby POU CRE. A model for the interaction and action of BmPOUM2 and BmAbd-A in regulation of the BmWCP4 expression is proposed.

Roundabout 1 (ROBO1)/SLIT2 Is a Novel Signaling Pathway in Multiple Myeloma Promoting Survival and Bone Marrow Niche Interaction

Roundabout 1 (ROBO1)/SLIT2 Is a Novel Signaling Pathway in Multiple Myeloma Promoting Survival and Bone Marrow Niche Interaction

Analysis of GATA1 Mutations in Down Syndrome Infants with Transient Abnormal Myelopoiesis and Clinical Impacts of GATA1 Mutation Types: A Report from the JPLSG TAM-10 Study

Introduction: Approximately 5-10% of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM). Almost all patients with TAM have GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). Although TAM patients exhibit various hematological abnormalities including circulating blasts, leukocytosis and thrombocytopenia, these abnormalities have been also reported in DS neonates without TAM. Therefore, analysis of GATA1 mutations is very important in the diagnosis of TAM. However, standard procedures to detect GATA1 mutations have not been established.Most of GATA1 mutations occur within the exon 2 and the surrounding sequences, but types of the mutations are varied, including insertions, deletions, duplications and point mutations. We previously reported that the expression levels of GATA1s were varied depending on types of mutations and might be associated with phenotypes of TAM including white blood cell (WBC) counts at diagnosis and a risk of progression to myeloid leukemia of DS (Kanezaki et al., Blood 2010). However, these findings have not been confirmed by other groups and effects of GATA1 mutation types on other clinical features of TAM have not been investigated.Patients and Methods: One hundred sixty-seven patients were enrolled in TAM-10 study and blood samples were available in 166 patients. GATA1 mutations were analyzed by Sanger sequencing using genomic DNA and complementary DNA (cDNA) prepared from peripheral blood. Expression patterns of GATA1 mRNA isoforms were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Targeted next-generation sequencing (NGS) were performed for patients in whom GATA1 mutations were not detected by Sanger sequencing. GATA1 mutations were classified into 3 groups according to the predicted consequences, splicing error (SE), loss of the first methionine (LOM) and premature termination codon (PTC). Blood smears were centrally reviewed. Patients whose smears were prepared more than 14 days after the onsets of TAM were excluded from the morphological analyses. Differences in clinical parameters among the 3 mutation groups were analyzed using the Fisher's exact test, Kruskal-Wallis test or Steel-Dwass test.Results: Mean age at sample collection, WBC count and blast percentage of blood samples were 8 days (range, 0-70 days), 22,100 µ/l (range, 4,400-422,000 µ/l), and 28.5% (range, 0-95%), respectively. GATA1 mutations were identified in 153 of 166 patients (92%) by Sanger sequencing. Although GATA1 mutations were not detected in 13 patients, splicing mutations were suspected in 7 patients because of the lack of the full-length GATA1 mRNA isoforms. In 12 of these 13 patients, blast percentages of the samples were less than 5%. GATA1 mutations were identified after targeted NGS in 10 of 13 patients negative for GATA1 mutations by Sanger sequencing. Of note, splicing mutations were confirmed after targeted NGS in all 7 patients suspected of having splicing mutations by RT-PCR. Collectively, GATA1 mutations were identified in 163 of 166 patients (98%). After exclusion of patients with multiple mutations (n=14) and internal deletion mutations (n=5), 39, 13 and 92 patients were classified into the SE, LOM and PTC groups, respectively. WBC counts at diagnosis were not significantly different among the 3 groups. However, the incidences of eosinophilia (>1,500 µ/l) were significantly different among the 3 groups (P<0.0001) and eosinophilia was more frequent in the SE (14/31, 45%) and LOM (4/11, 36%) groups than in the PTC (6/76, 8%) group (P<0.0001 and P=0.041, respectively). The levels of alanine aminotransferase (ALT) at diagnosis were also different among the 3 groups (P<0.00001) and the difference was statistically significant between the SE (median, 69; range, 11-468) and PTC group (median, 16; range, 3-380; P<0.00001).Conclusion: These results suggest that Sanger sequencing using cDNA as well as genomic DNA is rapid and sensitive method to detect GATA1 mutations and that targeted NGS is useful for detection of GATA1 mutations in patients with low blast percentages. GATA1 mutation types may affect some clinical features of TAM including the numbers of eosinophils and the levels of ALT. Because estimated expression levels of GATA1s are higher in SE and LOM groups than PTC group, high GATA1s expression might be associated with eosinophilia and increased levels of ALT in TAM. DisclosuresNo relevant conflicts of interest to declare.

Isoform Switch of TET1 Regulates DNA Demethylation and Mouse Development

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.

Construction of a draft reference transcripts of onion (Allium cepa) using long-read sequencing

To obtain intact and full-length RNA transcripts of onion (Allium cepa), long-read sequencing technology was first applied. Total RNAs extracted from four tissues; flowers, leaves, bulbs and roots, of red–purple and yellow-colored onions (A. cepa) were sequenced using long-read sequencing (RSII platform, P4-C2 chemistry). The 99,247 polished high-quality isoforms were produced by sequence correction processes of consensus calling, quality filtering, orientation verification, misread-nucleotide correction and dot-matrix view. The dot-matrix view was subsequently used to remove artificial inverted repeats (IRs), and resultantly 421 IRs were removed. The remaining 98,826 isoforms were condensed to 35,505 through the removal process of redundant isoforms. To assess the completeness of the 35,505 isoforms, the ratio of full-length isoforms, short-read mapping to the isoforms, and differentially expressed genes among the four tissues were analyzed along with the gene ontology across the tissues. As a result, the 35,505 isoforms were verified as a collection of isoforms with high completeness, and designated as draft reference transcripts (DRTs, ver 1.0) constructed by long-read sequencing.

The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis

The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis.

One amino acid deletion in collagen XVII-binding domain of plectin with a truncation mutation underlies epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a group of congenital skin fragility disorders characterized by intraepidermal skin detachment. PLEC, encoding plectin, is one of the causative genes responsible for EBS. Plectin deficiency is responsible for two autosomal recessive EBS subtypes: EBS with muscular dystrophy (EBS-MD) and EBS with pyloric atresia (EBS-PA). Plectin is a linker protein between hemidesmosomes and intermediate filaments. Plectin harbors a central rod domain which mediates homodimer formation. Plectin transcripts include both full-length and rodless forms. Previous studies indicate that loss of full-length plectin with the maintenance of rodless plectin leads to EBS-MD, whereas both full-length and rodless plectin isoforms are deficient in EBS-PA. However, a few EBS-MD cases have been reported to have in-frame deletions in the N-terminal globular domain of plectin. The Y domain of plectin (Arg618 to Val819) was implicated in binding to collagen XVII (COL17) in yeast two-hybrid assays. However the relevance of plectin-COL17 binding in EBS has not been uncovered. Here, we introduce an EBS patient harboring one amino acid deletion in the N-terminus globular domain (p.Phe755del) and a truncation mutation (p.Leu3065fs) in plectin. Plectin expression in skin specimens and cultured keratinocytes from the patient was diminished at the protein and the mRNA levels. COL17 expression was reduced at the protein level, whereas no pathogenic mutation was detected in the COL17A1 gene. Phe755, a deleted amino acid in one allele of the patient, is located in the Y domain of plectin (Arg618 to Val819). Our study is the first to demonstrate that defects of plectin-COL17 binding might contribute to the EBS phenotype.

Full-Length Isoform Sequencing Reveals Novel Transcripts and Substantial Transcriptional Overlaps in a Herpesvirus.

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

New Findings What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full‐length human OPN‐a isoform is the most pro‐inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD‐integrin‐dependent manner. OPN‐a upregulates expression of tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro‐inflammatory effects of OPN‐a, suggesting that a potential mechanism of OPN action is by promoting TNC–TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro‐inflammatory activities of human OPN isoforms (OPN‐a, OPN‐b and OPN‐c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN‐a > OPN‐b > OPN‐c) and dog muscle (OPN‐a = OPN‐c). In healthy human muscle, mechanical loading also upregulated OPN‐a expression (eightfold; P < 0.01), but did not significantly upregulate OPN‐c expression (twofold; P > 0.05). In vitro, OPN‐a displayed the most pronounced pro‐inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN‐a upregulated tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN‐mediated macrophage cytokine production. In summary, OPN‐a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN‐a promotes pro‐inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC–TLR4 signalling. Together, our findings suggest that specific targeting of OPN‐a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

The Intracellular Signaling Molecule Darpp-32 Is a Marker for Principal Neurons in the Cerebellum and Cerebellum-Like Circuits of Zebrafish.

The dopamine and cAMP regulated phosphoprotein of apparent molecular weight 32 kDa (Darpp-32) is an inhibitory subunit of protein phosphatase-1 (PP-1). Darpp-32 activity is regulated by multiple ligand-activated G-protein coupled receptors (GPCRs). This protein is coded for by the protein phosphatase-1 regulatory subunit 1b (ppp1r1b) gene. Here, we provide experimental evidence for the presence of multiple isoforms of ppp1r1b in zebrafish. We show that these isoforms are differentially expressed during development with the full-length isoform being maternally deposited. Next, with a custom polyclonal antibody generated against the full-length protein, we show that in the adult, Darpp-32 is strongly expressed in principal neurons of the cerebellum and cerebellum-like circuits. These include Purkinje neurons in the cerebellum, Type-I neurons in the optic tectum, and crest cells in the medial octavolateralis nucleus (MON). We confirmed the identity of these neurons through their colocalization with Parvalbumin 7 immunoreactivity. Darpp-32 is seen in the somata and dendrites of these neurons with faint staining in the axons. In all of these regions, Darpp-32-immunoreactive cells were in close proximity to tyrosine hydroxylase (TH) immunoreactive puncta indicating the presence of direct catecholaminergic input to these neurons. Darpp-32 immunoreactivity was seen in Purkinje neurons as early as 3 days post-fertilization (dpf) when Purkinje neurons are first specified. In sum, we show that Darpp-32, a signaling integrator, is a specific marker of principal neurons in the cerebellum and cerebellum-like circuits in zebrafish.

Abstract LB-014: Targeting insulin receptor splicing as a therapeutic intervention for pediatric rhabdomyosarcoma

Abstract The insulin receptor (IN-R) alternatively spliced variant A, encoding a receptor lacking exon 11, is the predominant isoform expressed in both pediatric sarcoma cells and vascular endothelial cells. Exon 11 of the IN-R gene is composed of 36 nucleotides that encode 12-amino acids that reside at the carboxyl terminus of the alpha subunit. Though only a small change in the protein composition, deletion of these 12 amino acids results in a receptor that has increased binding affinity for the IGF-2 growth factor and is exquisitely capable of responding to autocrine and paracrine signaling. Cells expressing both the IN-R A variant and the IGF-2 growth factor are therefore programmed for growth and angiogenesis, two essential steps in tumorigenesis. Furthermore, the IGF-2 can maintain angiogenesis through IN-R A when the IGF-1 receptor is blocked. We also demonstrate that hypoxia induces IN-R alternative splicing. This finding is particularly intriguing since recent studies have shown that adaptation to hypoxic environments is a hallmark of the neoplastic phenotype. We therefore hypothesize that the expression of splicing factors is altered by hypoxia leading to the generation of the IN-R A isoform, which contributes to cancer progression beyond the micrometastatic stage. To characterize sequence elements and RNA binding proteins in the regulation of IN-R alternative splicing, we have designed a hypoxia-induced splicing system that recapitulates the IN-R splicing seen in tumors. We have shown that sequence elements flanking the regulated exon are important for the hypoxia-induced splicing changes. Furthermore, have performed an antisense oligonucleotide (ASO) walk to target these splicing enhancer and repressor sequences to interfere with IN-R splicing. We have successfully targeted the CUGBP1 negative regulator of splicing that resides in intron 10 to increase the IN-R B full-length isoform. We have shown that an ASO used to drive production of the IN-R B isoform in a rhabdomyosarcoma (RMS) cell line, which normally expresses the IN-R A isoform, affects cancer cell behavior to induce reduced motility and cell migration. We therefore postulate that modulation of the splicing of the IN-R, alone or in conjunction with IGF-1R blocking antibodies, could be used as a therapeutic intervention in pediatric RMS cells. Citation Format: Dawn S. Chandler, Chelsea Brown, Hemant Bid, Thomas Bebee, Daniel F. Comiskey, Frank Rigo, Peter J. Houghton. Targeting insulin receptor splicing as a therapeutic intervention for pediatric rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-014.

Abstract 1200: HER-2 isoform interaction in mammary carcinoma onset and progression

Abstract Human breast cancer cells express full-length HER-2 along with proteins resulting from mutation, alternative splicing, alternative initiation of translation and post-translational modification. The Delta16 splice variant, lacking exon 16, has the properties of an activated oncogene, but it could also play beneficial roles in the response to targeted therapeutic agents. To study mammary carcinogenesis in a mouse model that mimics human coexpression of full-length HER-2 and Delta16 isoforms, we produced hybrid mice bearing heterozygous copies of both human transgenes (F1 mice), and we compared them to parental mice (Delta16 and HER-2 transgenic mice, respectively). Tumor onset in F1 and Delta16 mice was much faster than in HER-2 mice (30 vs &amp;gt;80 weeks), but the growth of established tumors and metastatic spread were not enhanced. Each mammary carcinoma of F1 mice expressed both isoforms at variable ratios. Most (∼80%) expressed high levels of Delta16 and low levels of full length HER-2, a few (∼5%) expressed full-length HER-2 and little, if any, Delta 16, and the remainder (∼15%) coexpressed at high level both isoforms. The study of tumor vascularization showed that full-length HER-2 tumors mainly contained few large vessels or vascular lacunae, whereas Delta16 tumors were perfused by numerous endothelium-lined small vessels. F1 tumors with high full-length HER-2 expression made few large vessels, whereas tumors with low full-length HER-2 and high Delta16 contained numerous small vessels and expressed high levels of both VEGF and VEGFR2. Administration of trastuzumab to young F1 mice effectively prevented mammary carcinoma onset in ∼85% of mice at 1 year of age. The preventive effect of trastuzumab was stronger in F1 mice than in both parental strain. To analyze the intrinsic sensitivity of F1 mammary carcinoma cells to targeted drugs in 3D, we established cell lines expressing different HER-2 isoform ratios. High Delta16 expression caused high sensitivity to HER-2 inhibitors (trastuzumab, neratinib, lapatinib), whereas high full-length HER-2 was associated with a lower sensitivity. Interestingly, high levels of both isoforms was associated with resistance to trastuzumab, but sensitivity to small molecule inhibitors. In conclusion, the coexpression of full-length HER-2 and Delta16 controls several determinants of mammary carcinoma development, progression and sensitivity to targeted agents, as revealed by the study of F1 mice. Citation Format: Arianna Palladini, Massimiliano Dall’Ora, Tania Balboni, Giordano Nicoletti, Marianna Ianzano, Roberta Laranga, Lorena Landuzzi, Veronica Giusti, Alessia Lamolinara, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni, Pier-Luigi Lollini. HER-2 isoform interaction in mammary carcinoma onset and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1200.

Abstract 733: Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5

Abstract Solid tumors exhibit a range of oxygen concentrations, in part due to abnormal vasculature and limited diffusion of oxygen which lead to hypoxia. Tumor hypoxia is associated with resistance to treatment, metastasis and poor patient outcome for many types of cancer. Assessing the impact of hypoxia in the tumor microenvironment and understanding how it contributes to tumorigenesis and survival is a challenging research goal for several reasons; a chief obstacle has been developing methods to analyze the transcriptome within precise regions of the tumor microenvironment in vivo. To address this task, we used Laser-Capture-Microdissection (LCM) of tumors labeled with the hypoxia probe, EF5, to specifically isolate normoxic and hypoxic regions of tumors from a murine model of head and neck cancer. We then carried out deep RNA-sequencing of the in vivo samples and corresponding in vitro samples grown in 0.5% oxygen conditions. Here we present the first described study of the hypoxic transcriptome in tumors at base-pair resolution. Our goal is to use these data to evaluate the potential of previously unstudied hypoxia-mediated pathways, such as the regulation of mRNA splicing, to be targeted therapeutically. We identified global changes in several classes of splicing in hypoxic compared to normoxic head and neck cancer cells, including changes in expression of last exons, retained introns and 3’ untranslated regions. Many of the alternatively spliced isoforms are in pathways central to hypoxic adaptation, such as glycolysis, cell cycle and translation. Among these genes, we uncovered a previously undescribed splicing event in the master regulator of translation, EIF2B5. The full-length protein encoded by EIF2B5 (eIF2Bå), is a necessary catalytic component of the eIF2B complex, which binds eIF2á and exchanges GDP for GTP to initiate translation. Strikingly, hypoxia induces retention of intron 12 in EIF2B5, which leads to insertion of an early stop codon in frame with the coding sequence; the resulting 65kDa protein isoform lacks a functional guanine nucleotide exchange (GEF) domain compared to the full-length isoform, but retains the region known to bind eIF2B complex subunits. Thus, we predict that the 65kDa isoform of eIF2Bå, may act in opposition to full-length eIF2Bå and inhibit translation initiation in conditions of oxygen deprivation. We are investigating EIF2B5, and other select hypoxia-regulated isoforms, to evaluate how alternative splicing impacts cell growth and survival in hypoxic tumors. Citation Format: Lauren K. Brady, Rohil Shekher, Vladimir Popov, Mircea Ivan, Milan Radovich, Constantinos Koumenis. Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 733.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

A survey of the sorghum transcriptome using single-molecule long reads

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.

Tumor suppressor p63 regulates Pirh2 ubiquitin ligase expression

Tumor suppressor protein p63 is a transcription factor of the p53 protein family. It is known that the proteins of this family, namely, p53, p63, and p73, perform a number of common tumor suppressor functions related to cell cycle regulation, apoptosis, and malignant transformation of cells, in addition to specific functions in the development of tissues and organs. All the proteins of the p53 family are regulated at post-translational level due to ubiquitination by E3 ligases and subsequent proteasomal degradation. The expression of the majority of E3 ubiquitin ligases, which modify the proteins of the p53 family, is regulated by the same proteins, thus forming negative feedback with them. In the present study, we first showed that the expression of the PIRH2 (RCHY1) gene, the product of which ubiquitinates all three members of the p53 family, is also regulated by p63. Regulation of the PIRH2 expression occurs at the transcription level by only fulllength TAp63 isoform, but not ΔNp63 isoform, which lacks a transactivation domain.

ApoE: In Vitro Studies of a Small Molecule Effector.

Apolipoprotein E4 (apoE4), one of three isoforms of apoE, is the major risk factor for developing late onset Alzheimer's disease. The only differences among these isoforms (apoE2, apoE3, and apoE4) are single amino acid changes. Yet these proteins are functionally very different. One approach to ameliorating the effect of apoE4 with respect to Alzheimer's disease would be to find small molecular weight compounds that affect the behavior of apoE4. Few studies of this approach have been carried out in part because there was no complete structure of any full-length apoE isoform until 2011. Here, we focus on one small molecular weight compound, EZ-482, and explore the effects of its binding to apoE. Using hydrogen-deuterium exchange, we determined that EZ-482 binds to the C-terminal domains of both apoE3 and apoE4. The binding to apoE4, however, is accompanied by a unique N-terminal allosteric effect. Using fluorescence methods, we determined an apparent dissociation constant of approximately 8 μM. Although EZ-482 binds to the C-terminal domain, it blocks heparin binding to the N-terminal domain. The residues of apoE that bind heparin are the same as those involved in apoE binding to LDL and LRP-1 receptors. The methods and the data presented here may serve as a template for future studies using small molecular weight compounds to modulate the behavior of apoE.

Molecular and geographic evolutionary support for the essential role of GIGANTEAa in soybean domestication of flowering time.

BackgroundFlowering time is a domestication trait of Glycine max and varies in soybeans, yet, a gene for flowering time variation has not been associated with soybean domestication. GIGANTEA (GI) is a major gene involved in the control of flowering time in Arabidopsis, although three GI homologs complicate this model in the soybean genome.ResultsIn the present work, we revealed that the geographic evolution of the GIGANTEAa (GIa) haplotypes in G. max (GmGIa) and Glycine soja (GsGIa). Three GIa haplotypes (H1, H2, and H3) were found among cultivated soybeans and their wild relatives, yet an additional 44 diverse haplotypes were observed in wild soybeans. H1 had a premature stop codon in the 10th exon, whereas the other haplotypes encoded full-length GIa protein isoforms. In both wild-type and cultivated soybeans, H2 was present in the Southern region of China, and H3 was restricted to areas near the Northeast region of China. H1 was genetically derived from H2, and it was dominant and widely distributed among cultivated soybeans, whereas in wild populations, the ortholog of this domesticated haplotype H1 was only found in Yellow River basin with a low frequency. Moreover, this mutated GIa haplotype significantly correlated with early flowering. We further determined that the differences in gene expression of the three GmGIa haplotypes were not correlated to flowering time variations in cultivated soybeans. However, only the truncated GmGIa H1 could partially rescue gi-2 Arabidopsis from delayed flowering in transgenic plants, whereas both GmGIa H2 and H3 haplotypes could significantly repress flowering in transgenic Arabidopsis with a wild-type background.ConclusionsThus, GmGIa haplotype diversification may have contributed to flowering time adaptation that facilitated the radiation of domesticated soybeans. In light of the evolution of the GIa gene, soybean domestication history for an early flowering phenotype is discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0653-9) contains supplementary material, which is available to authorized users.