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

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.