Exon 1B Research Articles

Ikaros isoforms: The saga continues

Through alternate splicing, the Ikaros gene produces multiple proteins. Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity. Ikaros isoforms interact to form dimers and potentially multimeric complexes. Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions. Small dominant-negative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros. Here, we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H. Although IK-1 is described as full-length Ikaros, IK-H is the longest Ikaros isoform. IK-H, which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4), is abundant in human but not murine hematopoietic cells. Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1. Moreover, IK-H can potentiate or inhibit the ability of IK-1 to bind DNA. IK-H binds to the regulatory regions of genes that are upregulated by Ikaros, but not genes that are repressed by Ikaros. Although IK-1 localizes to pericentromeric heterochromatin, IK-H can be found in both pericentromeric and non-pericentromeric locations. Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H, but not other Ikaros isoforms. The unique features of IK-H, its influence on Ikaros activity, and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

Identification and Characterization of the Alternatively Spliced Nuclear Receptor Coactivator-6 Isoforms

The nuclear receptor coactivator-6 (NCOA6, AIB3, PRIP, ASC-2, TRBP, RAP250 or NRC) is a co-activator for nuclear hormone receptors and certain other transcription factors. NCOA6 plays an important role in embryonic development, adipocyte differentiation, metabolism and breast carcinogenesis. The human and mouse NCOA6 genes had 15 and 14 previously identified exons, respectively. This study further identified an alternatively spliced exon 11b (E11b) in human or E10b in mouse, which codes a short polypeptide and a Stop codon, resulting in splicing variants lacking the last four exon-coded polypeptide. Analyses of mouse testis NCOA6 mRNAs identified four alternatively spliced variants, NCOA6-α (without E10b), -β (without E10a and E10b), -γ (with E10a and E10b) and -δ (without E10a but with E10b). These isoforms were detected in multiple mouse tissues and in MDA-MB-435 human cells. NCOA6-α and -β are mainly located in the nucleus; NCOA6-γ is located in both cytoplasm and nucleus; and NCOA6-δ is mainly located in mitochondria. The C-terminus coded by the last four exons was responsible for locating NCOA6-α and -β into the nucleus. The human E11a or mouse E10a-coded region is responsible for distributing NCOA6-γ in both cytoplasm and nucleus, while the region coded by E8-E9 in human or E7-E8 in mouse is responsible for directing NCOA6-δ to mitochondria. Our assays also demonstrated that NCOA6-α and -β could significantly enhance estrogen receptor α-mediated transcription, but NCOA6-γ and -δ were unable to do so. These results suggest that the diverse physiological function of NCOA6 may be mediated by multiple isoforms expressed in different tissues and localized in different subcellular compartments.

Isolation and phenotypic characterization of a multinucleated tartrate-resistant acid phosphatase–positive bone marrow macrophage

Prothrombin (PT) and osteopontin (OPN) promote adhesion of bone-derived tartrate-resistant acid phosphatase (TRAP, Acp5)-positive multinucleated cells differing in size, morphology, and resorptive activity. Here we explored phenotypic and functional differences between these cells. Global-wide and TRAP (Acp5) promoter messenger RNA expression, ability for phagocytosis, macrophage colony-stimulating factor-dependent migration, and in situ localization of these cells were investigated. Gene expression of PT-adherent cells was skewed toward expression of innate immune response, phagocytosis, and scavenger receptor genes. They avidly phagocytosed Staphylococcus aureus and Dextran particles, and their migration on PT was enhanced by macrophage colony-stimulating factor. In contrast, OPN-adherent cells lacked ability to phagocytose particles and their migration on OPN was inhibited by macrophage colony-stimulating factor. They expressed typical osteoclast proteases implicated in bone matrix degradation, such as the collagenases cathepsin K; matrix metalloprotease-2; -9; -13; and -14, consistent with their high bone resorptive activity in vitro. In addition, OPN-adherent cells predominantly expressed the PU.1/MiTF/NFATc1-driven TRAP exon 1C messenger RNA, whereas PT-adherent cells preferably expressed TRAP exon 1B messenger RNA. Furthermore, CD163/cathepsin-K immunohistochemistry demonstrated that PT-adherent cells were predominantly located in the diaphyseal bone marrow compartment, whereas the OPN-adherent cells were attached to cortical and distal metaphyseal trabecular bone surfaces. This study identifies differences in expression of several osteoclast and macrophage genes, as well as functional differences between PT- and OPN-adherent cells. We conclude that the OPN-adherent cells display osteoclast characteristics, especially with regard to expression of matrix-degrading enzymes, whereas the PT-adherent cell might represent a unique TRAP-positive multinucleated bone marrow macrophage implicated in immune recognition and phagocytosis.

B-raf Alternative Splicing Is Dispensable for Development but Required for Learning and Memory Associated with the Hippocampus in the Adult Mouse

The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

Alternative splicing of the antitrypsin gene in the silkworm, Bombyx mori

Alternative splicing plays an important role in expanding protein diversity. In the present study, different splice variants of the antitrypsin gene (sw-AT) in the silkworm were identified by bioinformatics analyses using expressed sequence tags and genomic information. Four splice variants were obtained by RT-PCR with suitably designed primers, confirmed by sequencing, and designated as sw-AT-1, sw-AT-2, sw-AT-3, and sw-AT-4. The sw-AT gene contains 10 exons and nine introns. The splice variants differ in exon 9, with sw-AT-1, sw-AT-2, and sw-AT-3 using different versions of the exon, namely exon 9a, 9b, and 9c, respectively. In sw-AT-4, exon 9 consists of the combination of exons 9b and 9c. The expression patterns of the four isoforms in different tissues, at different developmental stages, and under different stress conditions (temperature, starvation, and mycotic infection) were characterized and quantified. The sw-AT isoforms showed tissue-specific expression patterns, with sw-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. The four isoforms were predominantly expressed in the fat body, body wall, and testes of larvae, and exhibited similar expression profiles during development of the fat body. Among the stress treatments, low temperature had the greatest effect on isoform expression, and expression was also upregulated with mycotic infection.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

AbstractAbstract 815Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3‘ splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3‘ splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs.To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures:No relevant conflicts of interest to declare.

Human SHBG mRNA Translation Is Modulated by Alternative 5′-Non-Coding Exons 1A and 1B

BackgroundThe human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5′ UTR exons 1A and 1B differently regulate SHBG translation.ResultsCis-regulatory elements that could potentially regulate translation were identified within the 5′UTRs of SHBG TU-1A and TU–1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5′UTR sequence did show SHBG translation, whereas inclusion of the 5′UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5′UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.ConclusionsThe use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.

Tgfβ Signaling Directly Induces Arf Promoter Remodeling by a Mechanism Involving Smads 2/3 and p38 MAPK

We have investigated how the Arf gene product, p19(Arf), is activated by Tgfβ during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfβ signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfβ depend on Arf in vivo. Tgfβ1, -2, and -3 (but not BMP-2, another member of the Tgfβ superfamily) induce p19(Arf) expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in Arf(lacZ/lacZ) MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfβ type I receptor (TβrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfβ2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfβ2 effects, as does Cre recombinase treatment of Tgfbr2(fl/fl) MEFs to delete Tgfβ receptor II. Chromatin immunoprecipitation reveals that Tgfβ rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1β. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19(Arf) increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TβrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

Lower anxiety and a decrease in agonistic behaviour in Lsamp-deficient mice

In rodents, the Lsamp gene has been implicated in trait anxiety, fear reaction and fear conditioning. Human data link the LSAMP gene to several psychiatric disorders. In this study, we presented a general phenotypic characterization of Lsamp gene-deficient mouse line, created by deleting exon 1b. These mice displayed no gross sensory-motor deficiencies, no overt abnormalities and performed normally in memory and learning tests. However, they responded with increased activity to new environments. Moreover, they displayed reduced anxiety and notable deviations in social behaviour, such as lack of whisker trimming, reduced aggressiveness and reduced dominance. One possible explanation for the anxiolytic-like effect of the deletion of the Lsamp gene is a shift in balance in the Gabra1 and Gabra2 genes in the temporal lobe in favor of the Gabra2 transcript, encoding α2 subunit of GABA A receptors that mediate the stimulating effect of GABA agonists. The overall phenotype of Lsamp-deficient mice, characterized by decreased anxiety and several alterations in social behaviour, makes them a good model for studying the molecular mechanisms behind inadequate social behaviours observed in several psychiatric disorders.

CELF and PTB proteins modulate the inclusion of the β-tropomyosin exon 6B during myogenic differentiation

Exon 6B from the chicken β-tropomyosin pre-mRNA is alternatively spliced during myogenic differentiation. Exon 6B is excluded in mRNA from myoblasts and included in mRNA from myotubes. We investigated the regulation of exon 6B inclusion ex vivo in a quail myogenic cell line, which behaves as myoblasts in undifferentiated state and as myotubes after differentiation. We show that the β-tropomyosin exon 6B is a novel target of CUG-BP and ETR-3-like factor (CELF). Overexpression of CELF proteins in myoblasts activates splicing of exon 6B. Using a dominant-negative form of CELF4, we demonstrate that CELF proteins are involved in switching splicing from exon 6A towards exon 6B inclusion during myogenic differentiation. We also found that polypyrimidine tract binding protein (PTB) is required for splicing repression of exon 6B in myoblasts. CELF and PTB proteins exhibit antagonistic properties toward inclusion of exon 6B during myogenic differentiation. Our results suggest that a change in the protein level of CUGBP1 and PTB proteins, associated with a distinct pattern of PTB during the transition from myoblasts to myotubes is one of the parameters involved in regulating splicing of exon 6B during myogenesis.

HDMX-L Is Expressed from a Functional p53-responsive Promoter in the First Intron of the HDMX Gene and Participates in an Autoregulatory Feedback Loop to Control p53 Activity

The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.

Isoforms, Expression, Glycosylation, and Tissue Distribution of CTL2/SLC44A2

Antibodies to the solute carrier protein, CTL2/SLC44A2, cause hearing loss in animals, are frequently found in autoimmune hearing loss patients, and are implicated in transfusion-related acute lung injury. We cloned a novel CTL2/SLC44A2 isoform (CTL2 P1) from inner ear and identified an alternate upstream promoter and exon 1a encoding a protein of 704 amino acids which differs in the first 10-12 amino acids from the known exon 1b isoform (CTL2 P2; 706 amino acids). The expression of these CTL2/SLC44A2 isoforms, their posttranslational modifications in tissues and their localization in HEK293 cells expressing rHuCTL2/SLC44A2 were assessed. P1 and P2 isoforms with differing glycosylation are variably expressed in cochlea, tongue, heart, colon, lung, kidney, liver and spleen suggesting tissue specific differences that may influence function in each tissue. Because antibodies to CTL2/SLC44A2 have serious pathologic consequences, it is important to understand its distribution and modifications. Heterologous expression in X. laevis oocytes shows that while human CTL2-P1 does not transport choline, human CTL2-P2 exhibits detectable choline transport activity.

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Allele‐specific genetic interactions between Mitf and Kit affect melanocyte development

The tyrosine kinase receptor KIT and the transcription factor MITF, each required for melanocyte development, have been shown to interact functionally both in vitro and in vivo. In vitro, KIT signaling leads to MITF phosphorylation, affecting MITF activity and stability. In vivo, the presence of the Mitf (Mi-wh) allele exacerbates the spotting phenotype associated with heterozygosity for Kit mutations. Here, we show that among a series of other Mitf alleles, only the recessive Mitf (mi-bws) mimics the effect of Mitf (Mi-wh) on Kit. Intriguingly, Mitf (mi-bws) is characterized by a splice defect that leads to a reduction of RNAs containing MITF exon 2B which encodes serine-73, a serine phosphorylated upon KIT signaling. Nevertheless, other Mitf alleles that generally affect Mitf RNA levels, or carry a serine-73-to-alanine mutation that specifically reduces exon 2B-containing RNAs, do not show similar interactions with Kit in vivo. We conclude that the recessive Mitf (mi-bws) is a complex allele that can display a semi-dominant effect when present in a Kit-sensitized background. We suggest that human disease variability may equally be due to complex, allele-specific interactions between different genes.

Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1

BackgroundAlternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited.ResultsWe investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective.ConclusionsOur findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.

Glucocorticoid receptor auto-upregulation and its relation with glucocorticoid sensitivity in idiopathic nephrotic syndrome

Resistance to glucocorticoid (GC) treatment is a significant clinical problem in many diseases. Most effects of GC are mediated by glucocorticoid receptor (GR). Multiple promoters have been found in GR gene, which results in different exons 1s. In human leukemia cell lines, GC response is thought to be related with promoter usage and auto-upregulation of GR. In this study, we investigated whether GR could be auto-induced in peripheral blood mononuclear cells (PBMCs) and whether an inability to upregulate the GR is related with GC resistance in idiopathic nephrotic syndrome (INS). Reverse transcription-PCR was applied to measure the mRNA expression of GR transcripts (GR exons 1A, 1B, 1C, and GR), and real-time PCR was used to confirm these results. GR protein expression was analyzed by Western blot. Following GC treatment, both GR exon 1A and GR in PBMCs were increased in vitro, while GR exons 1B and 1C showed no significant changes. In patients with INS, the glucocorticoid-sensitive group expressed more GR exon 1A and less GR exon 1C than the glucocorticoid-resistant group, but the total GR showed no significant difference. GR is auto-induced in PBMCs in vitro and GR promoter 1A is involved in this mechanism; however the auto-upregulation of GR is not related with glucocorticoid response in patients with INS.

The Enamelin Genes in Lizard, Crocodile, and Frog and the Pseudogene in the Chicken Provide New Insights on Enamelin Evolution in Tetrapods

Enamelin (ENAM) has been shown to be a crucial protein for enamel formation and mineralization. Previous molecular analyses have indicated a probable origin early in vertebrate evolution, which is supported by the presence of enamel/enameloid tissues in early vertebrates. In contrast to these hypotheses, ENAM was only characterized in mammals. Our aims were to 1) look for ENAM in representatives of nonmammalian tetrapods, 2) search for a pseudogene in the chicken genome, and 3) see whether the new sequences could bring new information on ENAM evolution. Using in silico approach and polymerase chain reaction, we obtained and characterized the messenger RNA sequences of ENAM in a frog, a lizard, and a crocodile; the genomic DNA sequences of ENAM in a frog and a lizard; and the putative sequence of chicken ENAM pseudogene. The comparison with mammalian ENAM sequences has revealed 1) the presence of an additional coding exon, named exon 8b, in sauropsids and marsupials, 2) a simpler 5'-untranslated region in nonmammalian ENAMs, 3) many sequence variations in the large exons while there are a few conserved regions in small exons, and 4) 25 amino acids that have been conserved during 350 million years of tetrapod evolution and hence of crucial biological importance. The chicken pseudogene was identified in a region that was not expected when considering the gene synteny in mammals. Together with the location of lizard ENAM in a homologous region, this result indicates that enamel genes were probably translocated in an ancestor of the sauropsid lineage. This study supports the origin of ENAM earlier in vertebrate evolution, confirms that tooth loss in modern birds led to the invalidation of enamel genes, and adds information on the important role played by, for example, the phosphorylated serines and the glycosylated asparagines for correct ENAM functions.

Abstract 4933: Expression of the N-myc downstream-regulated gene 2 (NDRG2) is frequently suppressed by promoter hypermethylation in human gastrointestinal and pancreatic cancers

Abstract It is well known that inactivation of tumor suppressor gene (TSG) by promoter hypermethylation plays an important role during the development and progression of cancer. NDRG2 (N-myc downstream regulated gene 2) is considered to be one of the candidate TSGs. Significant down-regulation in various human cancers were observed, but the molecular mechanisms of reduced expression and the ways to carcinogenesis are largely not well understood. To elucidate the mechanisms in expressional suppression of NDRG2, we studied the methylation status of the putative promoter region in this gene. NDRG2 has two promoters; we first found that the mRNA transcription predominantly starts from the promoter containing exon 1b in organs of the digestive system. Then the expression level of NDRG2 was analyzed by quantitative real-time RT-PCR using 20 colorectal, 6 gastric, and 5 pancreatic cancer cell lines; many cell lines showed reduced expression. Bisulfite modification followed by nucleotide sequencing analyses in the putative promoter region revealed that the methylation status correlated with the suppressed expression. We extended our analysis to the primary tumors by immunohistochemical examination to elucidate clinical significance of suppressed expression of NDRG2 in tumorigenesis; matched normal and cancerous tissues from cancer patients with digestive system were analyzed and observed suppressed expression of NDRG2 in 19 of 20 colorectal, 8 of 10 gastric, and 85 of 86 pancreatic cancer specimens. We then designed a set of primer pairs for methylation specific PCR and examined the relationship between expression level of NDRG2 and promoter hypermethylation in resected primary tumor specimens. Association between methylation status and clinicopathologic features are now studied and the newly obtained results will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4933.

Abstract 1952: The rapid generation of mouse B cell lymphomas by lentiviral mediated overexpression of miR-1204 from a genetically unstable region of human 8q24

Abstract SNPs in the area surrounding MYC on human 8q24 have recently been found by Genome Wide Association (GWA) to be associated with susceptibility to a number of malignancies including prostate, breast, colorectal and bladder carcinomas (CA). This genetically unstable region is also a frequent target of chromosomal translocation (Tx), amplification or retroviral integration in a number of CAs such as breast, prostate, ovarian, colon, pancreatic, and cervical. In one example, essentially 100% of patients with Burkitt's lymphoma exhibit one of three characteristic non-random chromosomal Txs that places MYC or the immediate surrounding region in close proximity to enhancers of the immunoglobulin (Ig) heavy chain or light chain loci. Although de-regulated MYC expression could be assumed to be the target of the genomic instability or GWA-based susceptibility, no clear correlation between MYC expression and disease has been established. A possible alternative target has been identified in a series of transcripts cloned from the PVT1 region downstream of MYC. However, the extent of alternative splicing coupled with the lack of a coding region has made it difficult to assign a specific role for any PVT1-derived transcripts. Recently, we have identified a cluster of small RNAs exhibiting the hairpin formation, sequence conservation and expression characteristics of miRNAs (miR-1204∼1208) within the transcriptional domain of PVT1. Increased expression of several of these miRNAs in tumors harboring amplified MYC/PVT1 or Burkitt lymphomas with the 8q24 Tx suggests a possible role for these miRNAs in tumorigenesis, especially for miR-1204 which is found 60 kb downstream of MYC, flanking exon 1b of PVT1. An increased expression of miR-1204 in pre-B cells compared to pro-B cells also suggested a lymphoid specific developmental pattern of expression. Lentiviral constructs of miR-1204 under control of a CMV promoter (LentiCMV-miR-1204) revealed a possible effect on MYC, but in a pre-B (not pro-B) specific environment. Introduction of LentiCMV-miR-1204 into mice harboring either a MYC (C.iMYC) or IL6 (C.IL6) transgene resulted in high frequency and rapid onset of large B cell lymphomas (∼16 or 82 days vs. 91 or 117 days for controls, respectively). While these results point to over-expression of miR-1204 playing a specific role in the development of B cell malignancy, additional studies with the LentiCMV-miR-1204 construct in other tumor cell lines (prostate and breast) also reveal phenotypic changes in cell proliferation and migration. Further studies using lentiviruses with alternative promoters for miR-1204 and the other 8q24 associated miRNAs are underway to identify downstream targets and pathways not only in lymphoid malignancy but also in the wide range of malignancies associated with GWA-susceptibility and genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1952.