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Fontanari Krause, Luciana (2006): Identification and functional characterization of the human and murine OSTL gene, which encodes a RING-DRIL-RING domain protein possibly involved in B cell differentiation and leukemogenesis. Dissertation, LMU München: Faculty of Biology
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Abstract

The OSTL gene is localized at the band q23 in the chromosome 6. Its localization corresponds to a translocation breakpoint between chromosomes 6 and 12, the t(6;12)(q23;p13), that was characterized in our group in an acute lymphoblastic leukemia cell line. This translocation involves the ETV6 (translocation ETs leukemia) gene localized in chromosome 12 with the STL (six twelve leukemia gene) gene localized in chromosome 6. The STL gene shares the first exon with a novel gene, that we named OSTL (opposite STL), but they are transcribed in opposite directions. Since the fusion gene ETV6/STL encodes only for a very small protein which lacks any known functional domain, we speculate that the main leukemogenic effect of this translocation is the deregulation of OSTL. OSTL has a RING-Finger motif that is highly conserved between species and has a significant homology with other genes in human as well as C. elegans, D. melanogaster, and S. cerevisiae. OSTL showed a very specific expression pattern during the mouse embryogenesis. The aim of this project was the functional characterization of OSTL, with special emphasis in normal hematopoiesis and leukemogenesis. Therefore we have sequenced the whole human and mouse OSTL cDNA by using OSTL cDNA clones from the RZPD (“Resource Zentrum Primäre Datenbank”) in Berlin. These sequences encode for a 307 (mouse) and a 275 (human) amino acids length protein. The protein length differences between human and mouse are explained because of the existence of alternative spliced exons. The homology between human and mouse sequence is 99% at the protein level. The expression of GFP-OSTL fusion protein in mouse fibroblast cell line enable us to observe the subcellular localization of OSTL protein. GFP-OSTL is localized mainly in the cytoplasm, showing small spots, probably in the mitochondrial region. In a mouse multiple tissue Northern blot, we could show that OSTL is expressed in testis, ovary and liver. In an human multiple tissue Northern, OSTL expression was observed in skeletal muscle, testis, ovary, heart, placenta, pancreas and prostate. Northern blotting with different human cell lines revealed expression of OSTL in three EBV (Epstein Barr Virus) transformed lymphoblastoid cell lines (LCL B, LCL D, and LCL R) and in one NHL (Non-Hodgkin Lymphoma) cell line (Karpas 422). In Reverse Transcriptase PCR experiments using B cell in different maturation stages, the expression of OSTL was observed in naive, memory B and plasma cells, and in leukemic patient samples, expression was observed in several AML and ALL cDNAs. Whole mount in situ hybridization experiments were performed to investigate the temporo-spatial expression pattern of OSTL during mouse embryogenesis. There was distinct expression of Ostl in the somites (myotome), first and second branchial arches, optic and otic vesicles, in the hair follicles of the vibrissae, and limb buds in mouse embryos of embryonal days 9.5 to 14.5. This expression pattern suggests an important role for Ostl in the early development of these structures. Aiming to find protein interaction partners of OSTL, we performed a Yeast Two Hybrid assay using a Hela cDNA library. Among others we found interaction of OSTL with the antiapoptotic protein, HAX-1 (HS1-associated protein X-1), that is involved in the regulation of B-cell signal transduction, and interaction with the pro-apoptotic protein, SIVA. SIVA was originally identified as an interaction partner of CD27 (TNFRSF7), a member of the TNF-receptor superfamily, which is expressed in B cells. These interactions were confirmed by in vitro (cotransformation in yeast, CoIP) and in vivo (colocalization of these proteins in mammalian cells and CoIP) assays. Overexpression of Ostl in primary mouse hematopoietic cells followed by injection of the cells into lethally irradiated mice resulted in a T-Acute-Lymphoblastic-Leukemia (T-ALL) phenotype. In summary, our experiments could demonstrate that OSTL is important in B cell development and signaling and deregulation of this gene can contribute to the development of hematologic malignancies.