Toshiki Kameyama

Nested introns in an intron: evidence of multi-step splicing in a large intron of the human dystrophin pre-mRNA.

The mechanisms by which huge human introns are spliced out precisely are poorly understood. We analyzed large intron 7 (110 199 nucleotides) generated from the human dystrophin (DMD) pre‐mRNA by RT‐PCR. We identified branching between the authentic 5′ splice site and the branch point; however, the sequences far from the branch site were not detectable. This RT‐PCR product was resistant to exoribonuclease (RNase R) digestion, suggesting that the detected lariat intron has a closed loop structure but contains gaps in its sequence. Transient and concomitant generation of at least two branched fragments from nested introns within large intron 7 suggests internal nested splicing events before the ultimate splicing at the authentic 5′ and 3′ splice sites. Nested splicing events, which bring the authentic 5′ and 3′ splice sites into close proximity, could be one of the splicing mechanisms for the extremely large introns.

Spatiotemporal expression pattern of Myt/NZF family zinc finger transcription factors during mouse nervous system development

Three members of the Myt/NZF family of transcription factors are involved in many processes of vertebrate development. Several studies have reported that Myt1/NZF‐2 has a regulatory function in the development of cultured oligodendrocyte progenitors or in neuronal differentiation during Xenopus primary neurogenesis. However, little is known about the proper function of Myt/NZF family proteins during mammalian nervous system development. To assess the possible function of Myt/NZF transcription factors in mammalian neuronal differentiation, we determined the comparative spatial and temporal expression patterns of all three types of Myt/NZF family genes in the embryonic mouse nervous system using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. Results: All three Myt/NZF family genes were extensively expressed in developing mouse nervous tissues, and their expression was transient. NZF‐1 was expressed later in post‐mitotic neurons. NZF‐2 was initially expressed in neuronal cells a little earlier than NZF‐3. NZF‐3 was initially expressed in neuronal cells, just after proliferation was complete. Conclusion: These expression patterns suggest that the expression of NZF family genes is spatially and temporally regulated, and each Myt/NZF family gene may have a regulatory function in a specific phase during neuronal differentiation. Developmental Dynamics 243:588–600, 2014.

Myt/NZF family transcription factors regulate neuronal differentiation of P19 cells

During mammalian central nervous system development, neural stem cells differentiate and then mature into various types of neurons. Myelin transcription factor (Myt)/neural zinc finger (NZF) family proteins were first identified as myelin proteolipid protein promoter binding factors and were shown to be involved in oligodendrocyte development. In this study, we found that Myt/NZF family molecules were expressed during neuronal differentiation in vivo and in vitro. Transient over-expression of Myt/NZF family genes could convert undifferentiated P19 cells into neurons without induction by retinoic acid (RA), and the ability of these genes to induce neuronal differentiation was comparable to that of Neurog1 and Neurod1. Additionally, we found that St18 (or NZF-3) was induced by several bHLH transcription factors. When NZF-3 and Neurog1 were co-expressed in P19 cells, the rate of neuronal differentiation was significantly increased. These data suggest not only that NZF-3 works downstream of Neurog1 but also that it plays a crucial role together with Neurog1 in neuronal differentiation.

Fluvoxamine moderates reduced voluntary activity following chronic dexamethasone infusion in mice via recovery of BDNF signal cascades.

Major depression is a complex disorder characterized by genetic and environmental interactions. Selective serotonin reuptake inhibitors (SSRIs) effectively treat depression. Neurogenesis following chronic antidepressant treatment activates brain derived neurotrophic factor (BDNF) signaling. In this study, we analyzed the effects of the SSRI fluvoxamine (Flu) on locomotor activity and forced-swim behavior using chronic dexamethasone (cDEX) infusions in mice, which engenders depression-like behavior. Infusion of cDEX decreased body weight and produced a trend towards lower locomotor activity during darkness. In the forced-swim test, cDEX-mice exhibited increased immobility times compared with mice administered saline. Flu treatment reversed decreased locomotor activity and mitigated forced-swim test immobility. Real-time polymerase chain reactions using brain RNA samples yielded significantly lower BDNF mRNA levels in cDEX-mice compared with the saline group. Endoplasmic reticulum stress-associated X-box binding protein-1 (XBP1) gene expression was lower in cDEX-mice compared with the saline group. However, marked expression of the XBP1 gene was observed in cDEX-mice treated with Flu compared with mice given saline and untreated cDEX-mice. Expression of 5-HT2A and Sigma-1 receptors decreased after cDEX infusion compared with the saline group, and these decreases normalized to control levels upon Flu treatment. Our results indicate that the Flu moderates reductions in voluntary activity following chronic dexamethasone infusions in mice via recovery of BDNF signal cascades.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

ETV6‐LPXN fusion transcript generated by t(11;12)(q12.1;p13) in a patient with relapsing acute myeloid leukemia with NUP98‐HOXA9

ETV6, which encodes an ETS family transcription factor, is frequently rearranged in human leukemias. We show here that a patient with acute myeloid leukemia with t(7;11)(p15;p15) gained, at the time of relapse, t(11;12)(q12.1;p13) with a split ETV6 FISH signal. Using 3′‐RACE PCR analysis, we found that ETV6 was fused to LPXN at 11q12.1, which encodes leupaxin. ETV6‐LPXN, an in‐frame fusion between exon 4 of ETV6 and exon 2 of LPXN, did not transform the interleukin‐3‐dependent 32D myeloid cell line to cytokine independence; however, an enhanced proliferative response was observed when these cells were treated with G‐CSF without inhibition of granulocytic differentiation. The 32D and human leukemia cell lines each transduced with ETV6‐LPXN showed enhanced migration towards the chemokine CXCL12. We show here for the first time that LPXN is a fusion partner of ETV6 and present evidence indicating that ETV6‐LPXN plays a crucial role in leukemia progression through enhancing the response to G‐CSF and CXCL12.

NUP214-RAC1 and RAC1-COL12A1 Fusion in Complex Variant Translocations Involving Chromosomes 6, 7 and 9 in an Acute Myeloid Leukemia Case with DEK-NUP214

DEK-NUP214 gene fusion in acute myeloid leukemia (AML) is associated with poor prognosis. It is most often a sole translocation and more rarely observed as complex chromosomal forms. We describe an AML case with complex karyotype abnormalities involving chromosome bands 6p23, 6q13, 7p22, and 9q34. RNA sequencing analysis revealed that exon 17 of NUP214 (9q34) was fused to exon 2 of RAC1 (7p22). We also detected that the 5′-end of intron 1 of RAC1 was fused with the antisense strand of intron 5 of COL12A1 (6q13). RT-PCR analysis confirmed the expression of DEK-NUP214, NUP214-RAC1, RAC1-COL12A1, NUP214, and RAC1. These results suggest that the 5′- and 3′-ends of NUP214 from the breakpoint in the same locus were fused to RAC1 and DEK, respectively, and the 5′-end of RAC1 was fused to COL12A1. The reading frame of NUP214 was not matched with RAC1; however, high expression of the RAC1 protein was detected by Western blotting. This study identifies the variant complex fusion genesNUP214-RAC1 and RAC1- COL12A1 in a case of AML.

Rearrangement of VPS13B , a causative gene of Cohen syndrome, in a case of RUNX1 – RUNX1T1 leukemia with t(8;12;21)

Variant chromosomal translocations associated with t(8;21) are observed in 3–4% of acute myeloid leukemia (AML) cases with a RUNX1–RUNX1T1 fusion gene. However, the molecular events that occur in variants of t(8;21) are not well characterized. In the present study, we report genetic features of a variant three-way translocation of t(8;12;21)(q22;p11;q22) in a patient with AML. In this patient, leukemia cells lacked azurophilic granules, which does not correspond with the classic features of t(8;21). RNA-seq analysis revealed that TM7SF3 at 12p11 was fused to VPS13B at 8q22 and VPS13B to RUNX1, in addition to RUNX1–RUNX1T1. VPS13B was located near RUNX1T1 and both were localized at the same chromosomal bands. The reading frames of TM7SF3 and VPS13B did not match to those of VPS13B and RUNX1, respectively. Disruption of VPS13B causes Cohen syndrome, which presents intermittent neutropenia with a left-shifted granulopoiesis in the bone marrow. Disruption of VPS13B may thus cause the unusual features of RUNX1–RUNX1T1 leukemia. Our case indicates that rearrangement of VPS13B may be additional genetic events in variant t(8;21).

Transcriptional activation of platelet-derived growth factor receptor α and GS homeobox 2 resulting from E26 transformation-specific variant 6 translocation in a case of acute myeloid leukemia with t(4;12)(q12;p13).

Sir, E26 transformation-specific (ETS) variant 6 (ETV6) is a member of the ETS family of transcription factors and is frequently rearranged in human hematological malignancies. Most translocations involving ETV6 result in the generation of in-frame fusion genes with oncogenic properties. Others include out-of-frame and antisense orientation fusions, providing ectopic promoters [1–3]. The pathogenesis of leukemia associated with ETV6 translocation has been intensively investigated; however, there are many fusion genes in which the mechanism of the oncogenic effects of ETV6 rearrangements has not been completely revealed. A recurrent chromosomal translocation, t(4;12)(q11-q12;p13), has been reported to be observed in acute myeloid leukemia (AML), refractory anemia with excess of blasts (RAEB), and natural killer (NK) cell leukemia [4–7]. Although cysteine-rich hydrophobic domain 2 (CHIC2) is known as a fusion partner of ETV6 in t(4;12)(q12;p13), the pathogenic effect of CHIC2–ETV6 remains controversial [4]. Cool et al. [5] reported the positional effect of ETV6 leading to the overexpression of GS homeobox 2 (GSX2), which locates next to CHIC2 on 4q12 and has a transforming effect on NIH3T3 cells. A 59-year-old male was admitted to our hospital in July 2010 with a 2-year history of unknown fever, skin eruption, and muscle cramps. Complete blood count revealed a white blood cell count of 4.2 9 10/L, hemoglobin level of 97 g/L, platelet count of 164 9 10/L, and increased levels of interleukin-6. The patient was diagnosed with autoinflammatory syndrome. After a series of tocilizumab injections, the patient showed modest improvement in symptoms. Later, a complete blood count profile showed pancytopenia, and the patient was referred to our department in September 2012. Examination of an initial bone marrow aspirate (BMA) revealed a normocellular marrow with increased atypical cells (20%) and no significant dysplasia, which was negative for myeloperoxidase (POX), Sudan black B (SBB), and chloroacetate esterase (CAE). Flow cytometric (FCM) analysis revealed that the atypical cells were positive for CD34, CD117, and human leukocyte antigen (HLA)-DR and negative for CD13, CD33, CD3, CD19, and CD20. Chromosome analysis revealed 46, XY, del(6). At that point, it was difficult to make a diagnosis of the hematological malignancy. After the administration of prednisolone, the patient showed a significant improvement in symptoms. In January 2014, his white blood cell count unexpectedly increased to 44.2 9 10/L and a peripheral blood smear showed >50% of the nucleated cells to be leukemic blasts. A subsequent BMA examination revealed a hypercellular marrow with extensive infiltration of blasts, which were positive for POX and SBB and negative for CAE. In addition, FCM analysis revealed that immature cells were positive for CD13, CD33, CD34, CD117, and HLA-DR. The patient was diagnosed with AML. Chromosome analysis revealed 46, XY, t(4;12)(q12; p13) in all 20 cells examined. Subsequently, his condition deteriorated and he became intolerant to intensive chemotherapy. He died of leukemia progression. Fluorescence in situ hybridization (FISH) analysis of leukemia blasts showed a normal ETV6 signal and imbalanced split signals of ETV6, which indicates splitting of the 50-end of ETV6 (Figure 1a). We applied RNA-seq to analyze the molecular events of t(4;12)(q12;p13); in this case, we used the bone marrow sample from the advanced phase (Appendix S1). RNA-seq analysis revealed that exon 1 of ETV6 was fused to exon 2 of CHIC2 (ETV6–CHIC2) (Figure 1b,c). A long variant of ETV6–CHIC2, which includes a 141-bp intergenic region (IGR) sequence between GSX2 and CHIC2, was also observed. ETV6–CHIC2 did not appear to have oncogenic effects because it was an out-of-frame fusion. Reciprocal CHIC2–ETV6 was not detected. This fusion pattern is the same as in the cases that Silva et al. have reported [7]. High expression of GSX2 was observed by reverse-transcription polymerase chain reaction (RT-PCR) analysis and RNA-seq mapping analysis (Figure 2). These results suggested that the breakpoint of 4q12 lies in the IGR between GSX2 and CHIC2, and exon 1

Contents Vol. 146, 2015

Jacqueline Smith Division of Genetics and Genomics Roslin Institute, Roslin Midlothian EH25 9PS (UK) Tel. (+44) 131 527 4200 Fax (+44) 131 440 0434 E-mail: jacqueline.smith@bbsrc.ac.uk Plant cytogenetics and genomics Andreas Houben Institute of Plant Genetics and Crop Plant, Research (IPK) Corrents-Str. 3 Gatersleben, D–06466 (Germany) Tel. (+1) 785 532 2364; Fax (+1) 785 532 5692 E-mail: houben@ipk-gatersleben.de