EBF1-JAK2 inhibits the PAX5 function through physical interaction with PAX5 and kinase activity.

Gene aberrations of B-cell regulators and growth signal components such as the JAK-STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1-JAK2 (E-J). E-J caused constitutive activation of JAK-STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E-J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E-J with PAX5 and kinase activity of E-J were required for E-J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E-J-positive ALL cells, which suggests that E-J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B.

INTRODUCTION: Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. PATIENT AND METHODS: The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. RESULTS: A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. CONCLUSION: A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

Essential role of a carboxyl-terminal α-helix motif in the secretion of coagulation factor XI.

BACKGROUND: Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear. OBJECTIVE: To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient. PATIENT/METHODS: The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant. RESULTS: We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal-truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion. CONCLUSION: FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect resulting from aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.