Download Cloning of the mouse BTG3 gene and definition of a new

Leukemia (1997) 11, 370–375
 1997 Stockton Press All rights reserved 0887-6924/97 $12.00
Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG
family) involved in the negative control of the cell cycle
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F Guehenneux1, L Duret2, MB Callanan3, R Bouhas1, S Hayette4, C Berthet1, C Samarut1, R Rimokh1, AM Birot1, Q Wang5,
JP Magaud1,4 and JP Rouault1
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Unite INSERM 453 affiliee au CNRS, 5Unite d’Oncologie moleculaire, Centre Leon Berard, Lyon; 2Laboratoire BGBP-UMR CNRS 5558,
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UCLB, Villeurbanne; Groupe de recherches sur les lymphomes, Institut A Bonniot, La Tronche and; 4Laboratoire de Cytogenetique et
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Cytogenetique Moleculaire, Hopital Edouard Herriot, Lyon, France
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It is well known that loss of tumor suppressor genes and more
generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the
mouse BTG3 gene and show that its human counterpart maps
on chromosome 21. This evolutionarily conserved gene codes
for a 30 kDa protein and is expressed in most adult murine and
human tissues analyzed. However, we demonstrate that its
expression is cell cycle dependent and peaks at the end of the
G1 phase. This gene is homologous to the human BTG1, BTG2
and TOB genes which were demonstrated to act as inhibitors
of cell proliferation. Its description allowed us to define better
this seven gene family (the BTG gene family) at the structural
level and to speculate about its physiological role in normal
and tumoral cells. This family is mainly characterized by the
presence of two conserved domains (BTG boxes A and B) of
as yet undetermined function which are separated by a nonconserved 20–25 amino acid sequence.
Keywords: BTG 1, 2, 3; PC3; TIS21; TOB; antiproliferative gene
Introduction
Cell cycle progression is tightly controlled by the sequential
activation of proliferative and antiproliferative genes. Imbalance in this control system can lead to tumorigenesis. Initially,
most cancers were thought to arise through inappropriate activation of oncogenes. However, increasing evidence indicated
that loss of function of antiproliferative genes (tumor suppressor genes) represented a major route to tumor development. Extensive searches using diverse strategies have since
led to the identification of a number of antiproliferative genes
including for example P53 and RB1, and more recently the
(CDK) cyclin-dependent kinase inhibitors.1,2 However,
characterization of other physiologically important antiproliferative genes was also achieved using other cloning strategies (GAS genes, for instance).3
The molecular characterization of a chromosomal translocation observed in a lymphoid malignancy led us to clone a
gene, BTG1, which acts as an inhibitor of cell proliferation.4,5
Sequence analysis revealed that BTG1 was highly homologous to the rat PC3 and mouse TIS21 genes;5,6 PC3 being
expressed in the neuronal PC12 cell line induced to differentiate by the nerve growth factor7 and TIS21 expression
induced by serum stimulation of starved fibroblastic cell
lines.8 Recently, we cloned the human counterpart of the
PC3/TIS21 gene, BTG2. We demonstrated that BTG2
expression is regulated by P53, involved in the negative regulation of the cell cycle and in response to DNA damage
induced by different genotoxic agents.9 Recently, antiproliferative activity for PC3 was confirmed by another group.10
Finally, TOB, another gene homologous to BTG1 and 2, was
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Correspondence: J-P Rouault, INSERM U453, Centre Leon Berard, 28
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rue Laennec, F-69373 Lyon Cedex 08, France
Received 20 November 1996; accepted 11 December 1996
shown to be involved in the modulation of the biochemical
growth signal transduced by the growth factor receptor ErbB2
and to exert an antiproliferative activity on NIH3T3 cells.11
Furthermore, consistent with a presumptive antiproliferative
activity, a role for these genes in cellular differentiation is evident. BTG1 was implicated in myoblast differentiation12 and
expression of PC3 is an early event observed during the differentiation of the neuronal precursors in the mouse.13 It is likely
that BTG1, 2 and TOB constitute a novel family of functionally related genes involved in the regulation of cell
proliferation/differentiation. In this paper, we describe the
isolation/characterization of a new BTG member, the mouse
BTG3 gene. Structural analysis of this new gene reveals a new
common functional signature between the different members
of the BTG family. We speculate on its possible physiological
role in normal and tumoral cells.
Materials and methods
Cells and culture conditions
Peripheral blood lymphocytes (PBL) were obtained from the
blood of normal volunteers by centrifugation on triosil Ficoll. Mitogen-stimulated lymphocytes were obtained by incubating PBL at a concentration of 2 × 106 cells/ml, in the presence of phytohemagglutinin (PHA; Welcome, Dartford, UK)
diluted 1:200 in RPMI1640 medium supplemented with
20% fetal calf serum plus 0.03% L-glutamin, 100 mg/ml of
penicillin G and 100 mg/ml of streptomycin sulfate. BTG3
mRNA level were then assessed at various times after PHA
stimulation.
RNA isolation
Total cellular RNA was isolated from frozen samples by the
acid guanidium thiocyanate-phenol-chloroform method. For
Northern blot analysis, total RNA was size fractionated in
formaldehyde–1.2% agarose gels and transferred into nylon
filters. Probes a-32P-labeling, prehybridization, hybridization, and washing were carried out as previously
described.4
Sequencing procedures and sequence analysis
Overlapping deletions of DNA and cDNA cloned into
Bluescript SK(−) (Stratagene, La Jolla, CA, USA) were obtained
by the unidirectional exonuclease III digestion method (Erasea-base system; Promega, Madison, WI, USA). Deletion clones
were sized on agarose gels, and inserts of the selected clones
were sequenced by the dideoxy chain termination procedure
The BTG antiproliferative gene family
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F Guehenneux et al
with Sequenase II (USB, Cleveland, OH, USA) as described
by the manufacturer. Sequence similarity searches were
performed at the NCBI BLAST Web server (http://
www.ncbi.nlm.nih.gov/BLAST/) using the BLASTP, BLASTN
and BLASTX programs.14 Homologous sequences were
aligned with the CLUSTALW program15 and phylogenetic
trees were calculated according to the neighbour-joining
method.16
Preparation and analysis of DNA and cDNA libraries
A 10-day mouse embryo cDNA library cloned in lEXloxtm
was purchased from NOVAGEN (Madison, WI, USA) and processed according to the manufacturer’s instructions. Approximately 800 000 clones were screened with the BTG1 (RIA)
and TIS21 probes which were previously described.5–9
Hybridization of the filters was carried out using low stringency conditions (30% formamide, washing 3 × 20 mn at
room temperature in 2 × SSC, 0.5% SDS). Inserts from the
recombinant positive clones were characterized by partial
sequencing. The mouse BTG3 genomic counterpart was isolated by screening an EMBL3 phage mouse genomic DNA
library9 with a BTG3 cDNA probe.
In vitro translation assay
The BTG3 open reading frame (ORF) was subcloned into
bluescript (SK−) and in vitro transcribed following standard
procedures with T7 and T3 RNA pol (Boehringer, Mannheim,
Germany). Specific mRNA were then translated using the ‘reticulocyte type I’ in vitro translation kit, (Boehringer Mannheim
Biochemica) and the synthesized peptide was analyzed following the manufacturer’s instructions.
Human chromosomal localization
Human chromosome localization of the BTG3 gene was
determined by Southern blot analysis of a human × rodent cell
hybrid DNA panel purchased from BIOS laboratories
(Newhaven, CT, USA) with a mouse BTG3 ORF probe.
Results
Several recombinant phage clones containing inserts different
from BTG1 and TIS21 were isolated by low stringency screening of a 10-day mouse embryo cDNA library with BTG1 and
TIS21 32P-labelled probes. Partial sequencing of the clones
showed that they all code for the same protein. The corresponding gene was hence named BTG3. The longest clone –
1393 nucleotides (nt) – was sequenced on both strands as
described in Materials and methods. The sequence exhibited
an open reading frame coding for a peptide of 252 amino
acids (aa), predicted molecular weight 28981, and a 455-nt
long A + T rich 39 untranslated region. Finally, a polyadenylation site AATAAA (nt 1369) was found 25 bp upstream of the
poly-A tail (Figure 1). Screening of a mouse genomic DNA
library using a BTG3 cDNA probe and comparison of the
cDNA and genomic DNA sequences revealed that the BTG3
gene contained five exons with typical splice donor and
acceptor sites at the intron–exon junctions. The first exon is
rich in G + C nucleotides. The initiating codon of the longest
deduced ORF (nt 193–950, 252 aa) is localized at the beginning of the second exon and fulfills the criteria for the eukaryotic start signal with an adenine in position −3.17 In agreement
with the protein size predicted by sequence analysis, in vitro
translation of this ORF generated a protein with a 30 kDa
apparent molecular weight (Figure 2). Computer analysis of
the amino acid sequence (PROSITE program) did not reveal
any particular features except its homology with the other
BTG protein. Like the other members of this family, BTG3 is
evolutionarily conserved and zoo-blot analysis using a mouse
probe shows that sequences homologous to BTG3 could be
detected in human using normal stringency condition (data
not shown).
Northern blot analysis revealed that BTG3 expression is
ubiquitous, a specific 1.6 kb transcript (size in accordance
with the length of the cDNA) being detected with varying levels in most of the murine and human tissues analyzed (data
not shown), except in homogeneous non-proliferating cell
populations such as peripheral blood lymphocytes or serumstarved fibroblasts. The highest steady-state levels of the 1.6 kb
transcripts are observed in cell lines whatever their origin. Furthermore, unlike BTG1 and 25–9 which are expressed early in
the G1 phase of the cell cycle, it appeared that the highest
level of BTG3 transcript was observed only after 24–48 h of
stimulation of quiescent peripheral blood lymphocytes by
PHA at the end of the G1 phase of the cell cycle, CDK1 being
used as a marker of the G1–S transition18 (Figure 2).
Taking advantage of the conservation of the BTG3 gene during evolution, assignment of the BTG3 locus to the human
chromosome 21 was performed by Southern blot analysis of
a panel of interspecies (rodent × human) somatic hybrid DNA
using a mouse BTG3 cDNA probe (data not shown).
Similarity searches allowed us to identify several homologues from different vertebrate species. To clarify the
relationships between these genes, we calculated the multiple
alignment of homologous proteins and derived a phylogenetic
tree (Figure 3). To date, the BTG family comprises BTG1,
BTG2, BTG3, TOB, PC3, TIS21, TOB4, B9.10 and B9.15 (see
the accession numbers in Figure 3). During the preparation of
this manuscript, the cDNA sequences of the human and
mouse TOB5 genes have been deposited in databases
(Yoshida Y, Matuda S, Yamamoto T, 1996, unpublished; see
accession numbers in Figure 3). Sequence analysis showed
that BTG3 and TOB5 are a single gene. The phylogenetic tree
indicates that the human BTG2 gene is the counterpart of rat
PC3 and mouse TIS21 genes. The closest relatives of BTG3
are the xenopus B9.10 and B9.15 genes. However, the length
of the branch that links BTG3 to B9.10 and B9.15 suggests that
they are paralogous. Hence, the mammalian counterpart(s) of
xenopus B9.10 and B9.15 genes probably remain(s) to be
identified. In conclusion, the BTG family comprises at least
seven distinct genes in vertebrates: BTG1, BTG2 (TIS21, PC3),
BTG3/TOB5, TOB, TOB4, B9.10 and B9.15. Three groups of
proteins with similar lengths and specific conserved regions
can be distinguished in this family: (1) BTG3, B9.10 and B9.15
(233–252 aa); (2) BTG1 and BTG2 (158–171 aa); and (3) TOB
and TOB4 (344–360 aa) (Figure 4). Two short domains of
respectively 22 (box A) and 20 amino acids (box B) conserved
in all these proteins allowed us to define two signatures that
characterize the BTG family (Figure 4). These two domains,
separated by a spacer sequence of 20–25 non-conserved
amino acids, are located in the first 120 residues of the BTG
proteins.
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The BTG antiproliferative gene family
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F Guehenneux et al
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Figure 1
Nucleotide sequence of the mouse BTG3 cDNA (Genbank accession number Z72000). The standard one-letter code is used for
nucleotides and amino acids. The positions of the exons are indicated by arrows. The sequences of the conserved domains A and B are
underlined. Bold type ATG and AATAAA indicate the initiation and the polyadenylation sites.
The BTG antiproliferative gene family
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F Guehenneux et al
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Figure 2
(a) BTG3 expression after PHA stimulation of peripheral blood mononuclear cells. The Northern blot was successively hybridized
to BTG3 (1.6 kb), CDK1 (1.6 and 2 kb) and S26 (0.7 kb) probes. S26 is an internal control that ascertained the equal loading of RNA in each
lane.23 (b) SDS-PAGE analysis of the BTG3 ORF in vitro translation. +, sense BTG3 RNA; −, antisense BTG3 RNA.
Figure 3
Phylogenetic tree of all the proteins from the BTG family. Genbank accession numbers: BTG1: X61123 (human); L26268 (rat),
Z16410 (mouse), X64146 (chicken); BTG2/TIS/PC3: U72649 (human), M64292 (mouse), M60921 (rat); TOB: D38305 (human); B9.10; X73317;
B9.15: X73316; BTG3: Z72000 (mouse); TOB5: D83745 (mouse), D64110 (human); TOB4: D64109 (human).
Discussion
In this paper we report the cloning of the mouse BTG3 gene
and show that its human counterpart maps on chromosome
21. This evolutionarily conserved gene codes for a 30 kDa
protein and is expressed in most adult murine and human
tissues analyzed. However, we demonstrate that its expression
is related to the cell cycle in PHA-stimulated human peripheral blood lymphocytes, the highest levels of BTG3 transcripts
being observed at the end of the G1 phase of the cell cycle
with a peak at the G1–S transition. In quiescent PBL, BTG3
mRNA is undetectable. BTG3 is therefore confirmed as a new
member of the BTG family which already comprises at least
three members (BTG1, 2 and TOB) involved in growth control
and or differentiation. The most remarkable structural features
of the BTG family reside in the existence of two highly conserved short domains (BTG boxes A and B) separated by a
relatively constant (20–25 aa) stretch. This molecular organization is reminiscent to that of other proteins playing a major
role in cell growth control such as the pocket protein family
The BTG antiproliferative gene family
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F Guehenneux et al
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Figure 4
Comparison of the amino acid sequences of the predicted BTG protein family members. They were aligned with the CLUSTALW
program. The standard one-letter code is used for the amino acids.
(RB1, p107, p130) and the BCL2 protein family.19,20 It is likely
that, as described for the pocket protein family, the BTG boxes
are involved in protein–protein interactions. So far, it has been
demonstrated that TOB binds to and probably modulates the
ERB-2 receptor signal transduction11 and that BTG1 and TIS21
bind and activate a protein arginine methyl transferase
(PRMT1), some substrates of which have already been characterized (Histone/hnRNP A1).21
Another point to be discussed is the timing of expression of
BTG1, 2 and 3 during the cell cycle. In fact, BTG1 and 2 are
expressed in early G1 and BTG3, as demonstrated in the
present work, is expressed in late G1 before the entry of the
cells in S phase. Although we do not know the exact function
of the BTG/TOB family, we can assume that these genes control different checkpoints in G1 and could constitute the negative counterpart of the G1 cyclins which are also sequentially
activated during this phase.1,2 In this respect, the activity of
the BTG genes could be compared to that of the cyclin G1dependent-kinase inhibitors but we have never been able to
demonstrate interaction of any of these genes with CDK or
cyclins. The fact that BTG1 and 2 bind to and modulate the
enzymatic activity of PRMT suggests that methylation of target
proteins at different stages of the cell cycle may play a role
in cell growth and or differentiation control.21 So far, phosphorylation and dephosphorylation of various substrates have
been considered to play a central role in cell cycle regulation.
However, recent works strongly suggest that other post-translational modifications such as histone acetylation may also be
implicated in growth and differentiation control.22 Now, in the
light of our results, it remains to be demonstrated that enzymatic systems involved in the transfer of methyl groups
(protein methyltransferase and methylesterase) might participate in the regulation of homeostasis.
Goals for future studies include characterization of the BTG
proteins partners in order to define more precisely if they constitute the elements of a novel negative signalling pathway.
With regards to their growth inhibiting activity, these genes
could act as tumor suppressor genes and studies to find if they
are inactivated in some solid tumors or malignant blood disorders would be justified.
Acknowledgements
This work was supported by grants from ARC (1391) and Ligue
Nationale Contre le Cancer. FG is the recipient of a fellowship
from Ligue Nationale Contre le Cancer. We thank M Billaud
for helpful discussion.
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