Enforced expression of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity.. Our results show
Trang 1EVI1 and suppresses cell transformation
Roger McGilvray1, Mark Walker2and Chris Bartholomew1
1 Department of Biological & Biomedical Sciences, Glasgow Caledonian University, UK
2 CRUK Beatson Laboratories, Beatson Institute for Cancer Research, Glasgow, UK
EVI1 is a member of the PR domain family of
pro-teins [1], which include PRDM1 (PRDI-BF1) [2],
PRDM2 (RIZ) [3] and PRDM16 (MEL1) [4],
and share structural similarities with N-terminal PR
domains, functional similarities with transcriptional
repressor activities, have multiple cys2⁄ his2 zinc-finger
motifs, and roles in cell differentiation and
tumorigen-esis The complex phenotype observed in Evi1
knock-out mice suggests that it has a role in a range of
biological processes including general cell proliferation,
vascularization and cell-specific developmental
signal-ling [5] The pleiotropic phenotype reflects Evi1’s
func-tions as a transcription factor [6] as well as its ability
to interfere with several signalling pathways through interactions with Smad [7,8] and JNK [9] proteins The Evi1 transcriptional repressor protein is essen-tial for normal development [5] and when inappropri-ately expressed participates in the progression of a subset of leukaemias and myelodysplasias [10] In vitro studies have shown that a number of biological prop-erties can be attributed to the Evi1 protein including: (a) deregulation of cell proliferation [11]; (b) inhibition
of transforming growth factor-b, bone morphogenic protein and activin signalling [7,8]; and (c) inhibition
of stress-induced apoptosis [9] These activities require the DNA-binding domains ZF1⁄ ZF2 [12,13] and a
Keywords
ART-27; cell transformation; EVI1; leukemia;
ubiquitously expressed transcript
Correspondence
C Bartholomew, Glasgow Caledonian
University, Department of Biological &
Biomedical Sciences, City Campus,
Cowcaddens Road, Glasgow G4 OBA, UK
Fax: +44 (0)141 331 3208
Tel: +44 (0)141 331 3213
E-mail: c.bartholomew@gcal.ac.uk
(Received 30 June 2006, revised 10 May
2007, accepted 8 June 2007)
doi:10.1111/j.1742-4658.2007.05928.x
The EVI1 transcriptional repressor is critical to the normal development of
a variety of tissues and participates in the progression of acute myeloid leu-kaemias The repressor domain (Rp) was used to screen an adult human kidney yeast two-hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated Enforced expression
of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity The Rp-binding site for UXT was determined and non-UXT-binding Evi1 mutants (Evi1D706–707) were developed which retain the ability to bind the corepressor mCtBP2 Evi1D706–707 transforms Rat1 fibroblasts, show-ing that the interaction is not essential for Evi1-mediated cell transforma-tion However, Evi1D706–707 produces an increased proportion of large colonies relative to wild-type, showing that endogenous UXT has an inhibi-tory effect on Evi1 biological activity Exogenous UXT still suppresses Evi1D706–707-mediated cell transformation, indicating that it inhibits cell proliferation and⁄ or survival by both Evi1-dependent and Evi1-independ-ent mechanisms These observations are consistEvi1-independ-ent with the growth-suppressive function attributed to UXT in human prostate cancer Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1
Abbreviations
CtBP, C-terminal binding protein; DBD, DNA-binding domain; GST, glutathione S-transferase; Rp, repressor domain; SD, synthetic dropout medium; UXT, ubiquitously expressed transcript.
Trang 2200-amino acid transcriptional repressor domain
(Rp) [6]
Rp is critical to the biological activity of Evi1 We,
and others, have shown that Rp binds the C-terminal
binding protein (CtBP) family of corepressor proteins
that interact via two conserved PLDLS-like motifs
[14,15] Critically, the ability of Evi1 to repress
tran-scription, deregulate cell growth [14] and inhibit
trans-forming growth factor-b, bone morphogenic protein
and activin signalling [7,8,15], all rely on the
recruit-ment of CtBP
Several lines of evidence suggest that Rp is involved
in additional protein interactions that may also
con-tribute to Evi1’s broad spectrum of biological
activit-ies C-terminal deletion mutants of Rp, which retain
CtBP-binding sites, create more effective repressors [6],
suggesting that this activity is regulated Naturally
occurring splice variants in both human and murine
Evi1 exist that insert nine amino acids (FP⁄
QLPDQRTW) into Rp [16,17], which may either
create or disrupt novel or pre-existing interactions,
respectively Inspection of aligned human and murine
Rp primary amino-acid sequences with the
correspond-ing region of the related MEL1 protein [4] reveal
signi-ficant stretches of conservation in addition to the
CtBP-binding sites, suggesting additional common
activities might have been conserved during evolution
for these two proteins
To date, several Evi1-binding proteins have been
identified using a candidate protein approach It
remains possible that Evi1 interacts with as yet unknown cellular proteins responsible for regulating biological activity To investigate this, we screened a yeast two-hybrid library to identify new Rp-binding partners
Results
Isolation of a new Evi1 Rp-domain binding protein
Rp was subcloned from pGBT9Rp [14] into the kana-mycin-selectable yeast vector pKGI [18] to create pKGIRp (see Experimental procedures) As expected, pKGIRp produces a GAL4 DNA-binding domain (DBD)⁄ Rp protein which interacts with mCtBP2 in yeast AH109 cells, confirming its suitability for use
in screening a yeast two-hybrid library (Fig 1A–C; pKGIRp, pGAD10mCtBP2)
In total, 1· 106 independent clones from a human adult kidney yeast two-hybrid library (Clontech) were screened with pKGIRp in AH109 cells Initially, 37 potentially interacting clones were identified of which only 17 grew under more stringent conditions Target plasmid DNAs recovered from these clones were intro-duced into AH109 cells with pKGIRp and growth was reassessed Six recombinant plasmids contained genes encoding putative Rp-interacting proteins (Table 1, secondary screen) Sequencing of the plasmid DNA inserts revealed that five were HuCtBP2 (A6, A7, A10,
Fig 1 Interaction of Rp and A1 in yeast
cells (A,D) Yeast AH109 cells (Clontech)
were transformed with the combination of
plasmids shown The growth of single yeast
colonies containing these plasmids are
shown in (B) and (E) on SD lacking L
-histi-dine and adenine and (C) on SD plus L
-histi-dine and adenine.
Trang 3A14 and A37) The remaining clone, A1, contained a
novel gene This gene was isolated repeatedly from
additional independent library screens (data not
shown) and together with HuCtBP2 represents the
only Rp-interacting proteins identified in the human
adult kidney library
AH109 cells only grow under stringent conditions
when they contain both Rp and A1 expressed as
GAL4DBD and GAL4AD fusion proteins, respectively
(Fig 1A–C; pKGIRp, pGAD10 A1), showing that
these proteins interact To see if A1 and Rp interact
irrespective of their fusion partners, GAL4AD or
GAL4DBD, a domain swap was undertaken A1 and
Rp were inserted into pGBT9 (pGBT9 A1) and
pGADT7 (pGADT7 Rp), respectively, and shown to
still interact in AH109 cells (Fig 1A–C; pGBT9 A1,
pGADT7 Rp) The yeast two-hybrid assay also
revealed that A1 can homodimerize in AH109 cells
(Fig 1D,E; pGBT9 A1, pGAD10 A1) Furthermore,
the interaction of A1 with Rp is specific because A1
does not interact with laminin, p53 (Fig 1A–C;
pGAD10 A1) or mCtBP2 (Fig 1D,E; pGBT9 A1⁄
pGAD10mCtBP2)
A1 is identical to a novel gene called UXT
The sequence of A1 is shown in Fig 2A It consists of
a 546-nucleotide cDNA, which includes a partial
poly(A) tail There is an unbroken reading frame of
162 amino acids that is continuous with the vector
GAL4DBD and terminates with TGA (Fig 2A) at
nucleotide 487 Inspection of the sequence shows that
the first ATG (Fig 2A) fits the Kozak consensus for
translation initiation [19], suggesting that the gene
nor-mally encodes a putative protein of 157 amino acids
with a predicted molecular mass of 18.2 kDa
Interro-gation of the NCBI nucleotide database revealed the
identity of A1 with a novel gene designated UXT (AF092737) that encodes a 157-amino-acid protein (Fig 2A) A1 is subsequently referred to as UXT The tissue distribution of UXT was examined using northern blot analysis This shows that UXT produces
an abundant transcript of 750 bp in all tissues exam-ined, with the highest expression levels in heart, skel-etal muscle, pancreas, peripheral blood leukocytes, thyroid and lymph node (Fig 2B) The transcript size
is consistent with A1 being almost full length, allowing for an additional 39 5¢ nucleotides described for UXT and a 200 nucleotide poly(A) tail
The tissue distribution of UXT shows that it is expressed in the same tissues as Evi1, including lung, kidney, ovary and heart RT-PCR was performed to confirm that both genes are expressed simultaneously
in the same cells Evi1 is abundantly expressed in the leukaemia cell line DA-3 [20] and primary mouse embryo fibroblasts (MEFS), but not in lymphoma-derived monocytic U937 cells (Fig 2C) UXT is expressed in all cell types examined (Fig 2C), confirm-ing that transcripts for both genes coexist in cells in which Evi1 is expressed, including leukaemia cells where Evi1 has been activated and in cells where Evi1
is normally expressed such as fibroblasts
UXT binds full-length Evi1 UXT⁄ Evi1 binding was confirmed using a glutathi-one S-transferase (GST)-pull down assay GST–Rp and GST–UXT fusion proteins were expressed and purified from Escherichia coli strain pLysS cells using bacterial expression vectors (see Experimental procedures)
35S-Methionine-labelled in vitro-translated UXT and Evi1 proteins were produced using the expression vectors pCDNA3–UXT (Experimental procedures) and pRC⁄ CMV FL [6], respectively (Fig 3A) GST pull-down assays were performed with combinations of
Table 1 Isolation of Rp-interacting proteins in yeast The number of colonies obtained (clone A1 to A37), their growth on various selective media (SM), and their production of a- and b-galactosidase from both the primary and secondary library screening are shown ND, not done.
A20, A24, A25, A26, A29, A32, A33
Growth on SM -Trp ⁄ -Leu ⁄ -His ⁄ -Ala A1, A6, A7, A10, A11, A14, A15, A17
A1, A6, A7, A10, A14, A37 A20, A24, A25, A26, A29, A32, A33 A36, A37
Trang 4Fig 2 (A) Sequence of clone A1 The
nuc-leotide sequence of clone A1 is shown.
Below is the sequence of UXT (lower case)
and regions of identity are indicated by -.
Below the nucleotide sequence is shown
the primary amino acid sequence using the
single letter code The bold nucleotide and
amino acid sequence is partial pGBT9 GAL4
DBD vector nucleotide and amino acid
sequence Bold boxed nucleotide sequences
show the predicted translation initiation site
for UXT ⁄ clone A1 and the translation
termin-ation site The boxed amino acid sequence
represents translation of predicted 5¢ UXT
noncoding leader sequence that maintains
the reading frame of GAL4 AD and UXT (B)
Northern blot analysis of UXT expression.
Human MTNTMblots (Human, Human II and
Human III; Clontech) containing 2 lg per
lane of the indicated poly(A + ) RNAs were
hybridized to a 32 P-labelled UXT (A1) probe.
Filters were washed stringently, 0.1· NaCl ⁄
Cit, 0.1% SDS, 65 C and bands were
visu-alized by autoradiography (C) RT-PCR
analy-sis of total cellular RNA derived from the
indicated cells using human ⁄ mouse-specific
Evi1 (HME1 ⁄ HME2), Uxt (HMUXT5 ⁄
HMUXT3) and Gapdh (GAPDH5¢ ⁄ GAPDH3¢)
primers The expected size fragments for all
three genes: Evi1 (467 bp); Uxt (278 bp) and
Gapdh (451 bp) are indicated by arrows.
M indicates the 1kb hyperladder (Bioline).
Trang 5bacterially expressed and 35S-methionine-labelled
pro-teins and the results are shown in Fig 3 These
con-firm that UXT binds Rp (Fig 3B, lane 5) and
furthermore also interacts with full-length Evi1
(Fig 3B, lane 7) Formation of the complexes are
spe-cific as they do not occur with GST alone (Fig 5B,
lanes 2 and 3)
The UXT⁄ Evi1 interaction was also confirmed in
mammalian cells using coimmunoprecipitation UXT
was inserted inframe with three copies of the HA
epi-tope tag into the expression vector pcDNA3 to create
pcDNA33HAUXT (Experimental procedures) Cells
from the human embryonic kidney cell line BOSC-23
were transiently transfected with various combinations
of the C-myc epitope-tagged Evi1-encoding vector
pcDNA3EVI1myc (A Coyle & C Bartholomew,
unpub-lished) and pcDNA33HAUXT, and portions of the
cell extracts were examined either directly by western
blot analysis or immunoprecipitated prior to western
blotting Western blot analysis of whole-cell extracts
with either HA-specific a-12CA5 or C-myc-specific a-9E10 shows the expected size epitope-tagged 21 kDa UXT (HAUXT) and 145 kDa Evi1 (EVI1myc) proteins, respectively, in cells transfected with the corresponding expression vectors (Fig 3C) Immuno-precipitation of cell extracts and western blot analysis with a-myc reveals EVI1myc in cells transfected with pcDNA3EVI1myc, as expected (Fig 3; IP a-9E10) In addition, western blot analysis of a-myc-immunopre-cipitated cell extracts with a-HA shows HAUXT only
in those extracts that also contain EVI1myc, confirm-ing that these two proteins form a complex Examina-tion of the UXT–Evi1 interacExamina-tion at the endogenous level using the same method must await new reagents
to be developed
UXT suppresses Evi1-mediated transformation Next we investigated whether UXT has an effect on Evi1 biological activity by examining the impact of
Fig 3 (A) SDS–PAGE of in vitro translated products showing 35 S-labelled UXT (lane 1) and Evi1 (lane 2) (B) Analysis of GST pull-down assays by SDS–PAGE White box indicates bacterially derived GST protein, stippled box represents in vitro translated UXT and bacterially derived GST–UXT fusion protein Grey box represents bacterially derived GST–Rp fusion protein Evi1 zinc-finger domains and repressor domains are indicated by black and grey boxes, respectively Hatched box shows acidic domain (C) Interaction of Evi1 and UXT (A1) in mammalian cells Various combinations of pcDNA3Evi1myc (4 lg) and pcDNA33HAUXT (1 lg), indicated by +, were transiently transfected into BOSC-23 cells as described previously [2] One-tenth of whole-cell extracts were utilized directly for western blot analysis and the remainder was immunoprecipitated with a-myc (Santa Cruz Biotechnology, IP a-9E10) prior to western blot analysis Whole-cell extracts and immunoprecipitation a-9E10 extracts were resolved by SDS–PAGE (10%) and sequentially probed with a-haemagglutinin (Boehringer Mann-heim, MannMann-heim, Germany; WB a-12CA5) and a-myc (WB a-9E10) mAb Proteins were visualized by ECLTM(Amersham Pharmacia Biotech) Protein size was estimated by comparison with Full Range Rainbow TM molecular mass markers (Amersham Pharmacia Biotech, not shown) Evi1myc and HAUXT fusion proteins are indicated by arrows.
Trang 6UXT expression on Rat1 and RatFL
(Evi1-trans-formed cells) cell transformation The retroviral
expression vector p50MHAUXTzeo, containing UXT
fused inframe to a HA tag, was created Recombinant
retrovirus produced in BOSC-23 cells was used to
infect Rat1 and RatFL cells and cell populations
expressing UXT were selected Neither UXT
expres-sion in Rat1 cells nor empty vector controls were
transforming (Fig 4A, lanes 1,3,4), whereas Evi1 was
(Fig 4A, lane 2) However, enforced expression of
UXT reduced the number of transformed colonies
pro-duced by RatFL cells by 50% (Fig 4A, lane 5) The
empty vector control had no effect in RatFL cells
(Fig 4A, lane 5) Cell extracts prepared from the var-ious cell populations confirmed that they each produce the expected UXT HA-tagged fusion protein (Fig 4B) The effect of UXT on Evi1 transcriptional repressor activity was also examined but no significant changes were observed (data not shown)
An Evi1 Rp domain mutant lacking UXT-binding activity enhances Rat1 transformation activity The UXT-binding region of Rp was determined using
a series of deletion mutants (Experimental procedures) using the yeast two-hybrid assay Binding was retained when the Rp fragment (514–724) was deleted from the N-terminus to amino acid 634 (fragment 634–724), but lost upon C-terminal deletions between 715 and 695 (data not shown) A series of refined deletion mutants were created from the C-terminus of the 634–715 frag-ment to determine the minimum deletion required to lose UXT-binding activity Yeast two-hybrid assays revealed that Rp UXT binding is lost upon deletion
of amino acids 706–707 (Fig 5A–C, quadrant 7–9) Western blot analysis with a-GAL4DBD confirmed expression of equally abundant correct size proteins (fragments were subcloned from pGBT9 to pGBKT7 for this purpose; data not shown)
The ability of an Rp-deletion mutant lacking only amino acids 706 and 707 (Rp D706–707) to bind UXT and mCtBP2 was examined using the yeast two-hybrid assay Results confirm that this mutant is unable to bind UXT (Fig 5D–F, quadrant 2) but retains the ability to bind mCtBP2 (Fig 5D–F, quadrant 5) The transforming activity of Evi1 containing the UXT-binding mutant Rp domain was investigated in Rat1 cells The Rp domain of the previously described vector, p50MRpWTneo, which has identical trans-forming activity to p50M4.6neo [14], was substituted for RpD706–707 to create p50MRpD706–707neo and populations of Rat1 cells expressing this gene were selected and tested for transformation using a soft agar colony assay The results show that Evi1D706–707 gen-erates the same number of transformed Rat1 cell col-onies as wild-type Evi1 (data not shown) However, the mutant protein produces a higher proportion of larger colonies (Fig 6A) Figure 6B shows the percent-age of total colonies that are > 0.3 mm in diameter, generated from populations of Rat1 cells expressing either WT or mutant forms of Evi1 Approximately 14% of colonies generated by Evi1D706–707 are
> 0.3 mm in size, whereas only 3–4% of Evi1 trans-formed colonies achieve these dimensions
To see if enforced expression of UXT inhibits the transforming activity of Evi1D706–707, the
colony-Fig 4 (A) Colony formation of Rat1 and RatFL cell populations
infected with the indicated retroviral vectors Numbers were
deter-mined for colonies > 0.1 mm derived from plating 103cells Error
bars indicate the average number of colonies observed from three
independent assays Schematic representation of Evi1 and UXT
proteins are as described in the legend to Fig 3 The HA epitope
tag is shown as a striped box (B) Western blot analysis with
a-haemagglutinin (as described in Fig 3) of whole-cell extracts
derived from Rat1 cells (1), p50MHAUXTzeo infected BOSC 23 (2),
Rat1 (3) and RatFL (4) cells The HAUXT fusion proteins are
indica-ted by an arrow.
Trang 7forming activity of Rat1 cells expressing both
exo-genous proteins was examined Populations of Rat1
cells infected with both p50MRpD706–707neo and
p50MHAUXTzeo were selected and the production of
macroscopic colonies in soft agar assessed The results
show that transformation is suppressed by 50% in
cells containing Evi1D706–707 and UXT (Fig 6C)
The empty vector control, p50MXZEO, has no effect
on Evi1D706–707 transforming activity
Discussion
We have shown that Evi1 can interact with at least
two proteins present in human adult kidney cells via
the Rp domain One interaction is with the human
homologue of mCtBP2, HuCtBP2, and its significance
has already been documented [14] Surprisingly, our
library screens did not isolate HuCtBP1, which
recog-nizes and binds the same conserved PLDLS motif as
HuCtBP2, despite PCR analysis of the human kidney
library demonstrating that HuCtBP1 was present (data
not shown) Evi1 has previously been shown to
associ-ate with both HuCtBP1 [21] and mCtBP1 (E Ritchie
and C Bartholomew, unpublished) The most likely
explanation is that no appropriate HuCtBP1 clones in
the library are expressed inframe with GAL4AD
Evi1’s interactions with UXT, an 18.2 kDa protein,
have not been described previously Enforced UXT
expression in RatFL cells moderately suppresses cell
transformation The Evi1 UXT-binding mutant
(Evi1D706–707) retains the ability to bind mCtBP2
and Rat1 cell-transformation activity is enhanced This
shows that UXT binding: (a) is not required for Evi1 interaction with mCtBP2; (b) is not required for Evi1-mediated cell transformation; and (c) has an inhibitory effect on Evi1 biological activity Interestingly, enforced expression of UXT with Evi1D706–707 in Rat1 cells still moderately suppresses cell transforma-tion The data suggest that at least part of the suppres-sor activity is mediated by its interaction with UXT but that enforced UXT expression has a general growth-suppressive activity that is independent of Evi1 In this regard, it would be interesting to see if UXT expression suppresses cell transformation by other oncogenes too
Recent studies suggest that Evi1 is a survival factor
It is able to protect cells against chemically induced apoptosis [22] and promote survival of haemopoietic stem cells [23] Therefore, negative regulation of Evi1 biological activity might compromise its survival func-tion, reducing cell transformation and⁄ or proliferation
by decreasing the ability of cells to proliferate
optimal-ly in new environments, for example, the anchorage-independent growth of Rat1 fibroblasts in soft agar displayed in the transformation assay
UXT is located on human chromosome Xp11 and was originally identified when searching for the X-linked genes responsible for Renpenning syndrome, Prieto syndrome and Sutherland–Haan syndrome that map to this region, although it does not appear to be involved in their development [24] As shown here, UXT is abundantly and ubiquitously expressed in all tissues examined Based on an EST database search it has been suggested that UXT is overexpressed in
Fig 5 Interaction of UXT deletion mutants with Rp and mCtBP2 in yeast cells (A,D) Yeast AH109 cells were transformed with the combination of plasmids shown The growth of single yeast colonies containing these plasmids are shown in (B) and (E) on
SD plus L -histidine and adenine and (C) and (F) on SD lacking L -histidine and adenine.
Trang 8tumour cells [24] and might have a role in
tumorigen-esis In this regard, it is interesting to note that acute
basophilic leukaemia is associated with translocation
t(X,6)(p11,q23) [25] and therefore UXT itself is a
can-didate for the disease-associated Xp11 gene or
alternat-ively its ubiquitously active promoter may deregulate
expression of a novel leukaemia gene located on 6q23
The function of UXT is not known, but it has
previ-ously been shown to associate with several other
pro-teins, each of which has a role in the regulation of
gene transcription These include the leucine-rich
pen-tatricopeptide repeat-motif-containing (LRPPRC)
pro-tein [26] and the transcriptional coactivator CITED2
[27] UXT is also known as ART-27, a transcriptional
coactivator that interacts with the N-terminus of the
androgen receptor [28] Interestingly, enforced
expres-sion of UXT⁄ ART-27 in LNCaP prostate cancer cells
inhibits proliferation and furthermore its expression is
downregulated in human prostate cancer [29] This
proliferation-suppressive activity is consistent with the
inhibition of Evi1-mediated Rat1 cell transformation
mediated by UXT in our studies Both these
experi-mental observations are in direct contradiction to the
earlier interpretation derived from interrogation of the
EST database [24] Furthermore, UXT expression has
also been shown to be elevated in bladder, breast,
ovary and thyroid tumours suggesting it may be a
tumour marker [30] This apparent discrepancy may be
reconciled if UXT effects on cell transformation are tissue specific or that either up- or downregulation contributes to oncogenesis Alternatively, there are at least two naturally occurring UXT splice variants, transcript variant 1 encoding a 157-amino acid protein (NM153477) and transcript variant 2 encoding an N-terminal truncated protein of 145 amino acids (NM004182), which might have opposing effects on cell transformation In this regard, it will be interesting
to investigate both the form of UXT and the structural integrity of the gene coding sequences in tumour cells where its expression is elevated
Several lines of evidence indicate that UXT regulates cell proliferation UXT is a target gene for the E2F family of transcription factors that regulate transition through the G1⁄ S phase boundary of the cell cycle [31] Both E2F1 and E2F6 inhibit UXT gene expres-sion [31,32] Furthermore, E2F6 corepresses UXT and other genes with common functions in tumour sup-pression, suggesting that it might have a similar activ-ity [32], consistent with its abilactiv-ity to inhibit cell transformation
UXT has also been isolated as STAP1 and classified
as a member of the a class prefoldin family [33] UXT (STAP1) is a component of a large protein complex that can regulate transcription in HeLa cells, consistent with its interaction with the Evi1 transcription factor observed here UXT has also been shown to be located
Fig 6 (A) Photograph of colonies formed
by Rat1 cell populations expressing the
indicated Evi1 proteins using a Leica GZ6
microscope (B) Histogram showing the
percentage of soft agar colonies > 0.3 mm
generated by populations of Rat1 cells
expressing either wild-type (Evi1) or mutant
(Evi1D706–707) Evi1 The percentage was
determined by counting total number of
col-onies generated and the number of colcol-onies
> 0.3 mm Colony numbers were
deter-mined from two independent experiments
(1 & 2) Control 1 and 2 are parental Rat1
cells Error bars indicate variation between
three independent assays for each
experi-ment (C) Histogram showing total number
of soft agar colonies > 0.3 mm produced
per 1000 Rat1 cells expressing the indicated
proteins Control is empty p50MXZEO
retro-viral vector Error bars indicate the average
number of colonies observed from three
independent assays No colonies are
observed in Rat1 cells in the absence of
Evi1 (not shown).
Trang 9in centrosomes and its overexpression disrupts
centro-some structure in human U2OS cells [30] It has been
suggested that UXT abnormality may cause
dysfunc-tion of the centrosome contributing to malignant
transformation [30] Centrosomes play a key role in
cell proliferation, serving both to nucleate polarized
microtubule arrays for mitotic spindle organization
and cytokinesis and providing a multiplatform scaffold
with protein-docking sites for integrating cellular
regu-latory events [34] This raises the possibility that Evi1
might contribute to the regulation of cell proliferation
by interacting with a component of centrosomes
Our results show that UXT inhibits Evi1-mediated
cell transformation in addition to the previously
des-cribed inhibition of cell-proliferation activity and
therefore may be a negative regulator of cell growth
UXT interacts with Evi1 and may be a direct negative
regulator of its biological activity The precise
molecu-lar mechanism by which UXT reduces Evi1 activity is
unknown UXT might mediate its negative control of
cell proliferation and transformation by directly
inter-acting with and regulating the activity of factors that
stimulate this biological activity such as Evi1 In
addition, UXT mediates its negative activity on cell
proliferation and transformation indirectly, by an
independent mechanism
Experimental procedures
Construction of plasmids
pKGIRp was created by inserting a EcoRI⁄ BamHI Rp
domain fragment from pGBT9Rp [14] into pKG1 [18]
pGBT9A1 was created by excising A1 as a BglII fragment
from pGAD10 A1and inserting into a BamHI site of
pGBT9 pGADT7Rp was created by excising a EcoRI⁄
BamHI Rp fragment from pKG1Rp and inserting into the
corresponding site of pGADT7 (Clontech, Mountain View,
CA) pGEXUXT was created by PCR amplification with
5¢- and 3¢-oligonucleotides CGCTGGATCCCGGGAGG
AGCCCATCATG and GGAAGAATTCTCAAATTCCA
GGAAAAAACCA, respectively, and insertion of UXT
fragments into BamHI⁄ EcoRI of pGEX2 (Promega,
Madi-son, WI) pGEXRp was created by PCR amplification with
5¢- and 3¢-oligonucleotides AAGCGGATCCCGCATTCT
CTCAATCAATG and AAGCTGAATTCGTAGCGCTC
TTTCCCCT and insertion of Rp fragments into BamHI⁄
EcoRI of pGEX1 (Promega) pcDNA3UXT was created by
inserting a HindIII⁄ BamHI UXT PCR fragment amplified
from pGAD10 A1 with oligonucleotides AATTCAA
GCTTGCGCAATGAAGGTGAAGG and AATTCGGAT
CCTCAATGGTGAGGCTTCTC pcDNA33HAUXT was
created by simultaneous ligation of a NcoI⁄ NotI-digested
UXT PCR fragment, amplified from pGAD10 A1 with 5¢- and 3¢-oligonucleotides GAATCCATGGCGACGAC GCCCCCTAAGCG and GAATTGCGGCCGCCTCAAT GTGAGGCTTC, respectively, with a NcoI⁄ EcoRI frag-ment containing three copies of the HA epitope from S3H-ERK2 (gift from W Kolch, Beatson Institute, Glasgow, UK) and EcoRI⁄ NotI-digested pcDNA3 (Invitrogen, Carls-bad, CA) p50MHAUXTzeo was created by PCR amplifi-cation of pGAD10 A1 with 5¢- and 3¢-oligonucleotides AGCTTGCGGCCGCATCATGTACCCATACGATGTTC CAGATTACGCTGCGACCCCCCTAAGCG and GCTG AATTCTCAATGGTGAGGCTTC which was digested with NotI⁄ EcoRI and inserted into the corresponding site
of p50Mxzeo [14] Rp domain deletion mutants were cre-ated by inserting EcoRI⁄ NotI-digested PCR fragments gen-erated using the following 5¢-oligonucleotide (E634)
3¢-oligonucleotides (N724) AATTGCGGCCGCTCAGTA GCGCTCTTTCCCCTT (N715) AATTGCGGCCGCTCA GTTCTCTGGCAGGGTGTT or EcoRI⁄ BamHI-digested PCR fragments generated with 3¢-oligonucleotides (B713) AGCTTGGATCCTATGGCAGGGTGTTGGGAGGAGC, (B711) AGCTTGGATCCTAGGTGTTGGGAGGAGCTC
GGAAGCTGAA (B707) AGCTTGGATCCTAAGCTCG GAAGCTGAACATGGA (B705) AGCTTGGATCCTA GAAGCTGAACATGGAGGGCAC, into EcoRI⁄ NotI-digested pGBT9N [14] or EcoRI⁄ BamHI-digested pGBT9 pGBT9RpD706–707 was created by site-directed mutagen-esis (QuickChange XL system, Stratagene, La Jolla, CA) of pGBT9Rp [14] with oligonucleotides 5¢-CCCTCCATGTT
GGTGTTGGGAGGGAAGCTGAACATGGAGGG The same primers were used for site directed mutagenesis of p50MRpWTneo [14] to create p50MRpWTD706–707neo
Cell culture, transfections, CAT and b-galactosidase assays
RatFL cells have been described previously [11] Rat1,
Rat-FL, Bosc-23, HEK293 and primary mouse embryo fibro-blasts were all maintained in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, sodium pyruvate and glutamine U937 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, sodium pyruvate and glutamine DA-3 cells were similarly maintained in the pres-ence of 10% WEHI-3-conditioned medium Procedures for transfections, production of helper-free recombinant retrovi-rus, retroviral infections and growth in soft agar, CAT and b-galactosidase assays have all been described previously [6] Cells infected with zeocin containing retroviral vectors were selected and maintained in 1 mgÆmL)1 zeocinTM (Invitro-gen)
Trang 10Yeast two-hybrid assay
For the primary screen, AH109 competent cells were
pre-pared and transformed as described by the suppliers
(Clontech) with 1 mg pKGIRp and 0.8 mg human kidney
cDNA plasmid library (Clontech) Total numbers of
trans-formed colonies were estimated by growing an aliquot of
transformed cells for 3 days, at 30C on synthetic dropout
medium (SD), 0.67% yeast nitrogen base without amino
acids (Difco Laboratories, Sparks, MD), 0.06%
CSM-HIS-LEU-TRP (Bio101, Inc., Irvine, CA), 2% glucose, pH 5.8,
1.5% select agar (Life Technologies, Grand Island, NY)
and 20 lgÆmL)1 l-histidine HCl (Sigma, St Louis, MO;
H-9511) The remaining cells were grown for 14 days on
SD without l-histidine Growth of any colonies was
subse-quently examined under stringent conditions on SD lacking
both l-histidine and adenine by substituting CSM for DO
supplement (-ADE,-HIS,-LEU,-TRP; Clontech)
Plasmid DNA was isolated from yeast colonies by
scraping into 1 mL TE buffer, pelleting cells and
resus-pending them in 0.5 mL 10 mm K2HPO4 pH 7.2, 10 mm
EDTA, 50 mm 2-mercaptoethanol, 0.25 mgÆmL)1
zymo-lase, at 37C for 30 min, mixing with 0.1 mL 25 mm
Tris⁄ HCl pH 7.5, 25 mm EDTA, 2.5% SDS, at 65 C for
30 min followed by 10 min at 0C with 166 lL 3 m
KOAc E coli strain DH5a electroporation-competent
cells were prepared according to Sambrook et al [35],
electroporated as described by Clontech and transformed
colonies selected on Luria–Bertani plates containing
50 lgÆmL)1 ampicillin Plasmid DNAs were prepared
using a NucleoSpin plus miniprep plasmid extraction kit
(Clontech) a- and b-galactosidase assays, respectively,
were performed as described by Clontech
GST pull-down assay
Bacterial cultures containing pGEX expression vectors were
induced with 1 mm isopropylthio-b-d-galactoside for 3 h
and cells were resuspended and sonicated in NETN buffer
(20 mm Tris pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5%
NP40) Extracts were cleared in a microfuge at 4C and
GST-fusion proteins bound to glutathione Sepharose by
mixing with glutathione Sepharose slurry equilibrated with
NETN at 20C for 30 min followed by centrifugation and
washing three times in NETN.35S-Labelled in vitro
transla-ted proteins were produced using TNT-coupled reticulocyte
lysates (Promega) GST pull-down assays were performed
by incubating GST-fusion protein⁄ glutathione Sepharose
conjugate (5 mg), 100 mg E coli cell extract and in vitro
translated protein in NETN, 4C, overnight Extracts were
washed three times in excess NETN, 1· in excess MTPBS
(150 mm NaCl, 16 mm Na2HPO4, 4 mm NaH2PO4, pH 7.3)
and bound protein eluted from complex in 50 mm
Tris⁄ 5 mm reduced glutathione
Immunoprecipitation
Cells were scraped into 0.25 mL of immunoprecipitation buffer [36], rapidly frozen, thawed at 0C for 1 h, then microfuged at 11 000 g for 10 min at 4C Supernatant was removed, and 25 lL was aliquoted as whole-cell extract for western blotting and the remainder incubated o⁄ n with anti-(c-myc a-9E10) serum (Santa Cruz Biotechnology, Santa Cruz, CA) at 4C and subsequently incubated with
50 lL of 50% slurry of rabbit anti-(mouse IgG)-coated protein A Sepharose beads for 2 h at 4C Beads were washed three times in immunoprecipitation buffer and pre-pared for western blot analysis
RT-PCR
Total RNA (0.2 lg, prepared using the RNazolTMB method) was amplified using the CalypsoTM RT-PCR system (BioGene Ltd., Kimbolton, UK) according to the manufac-turers instructions The coupled reaction was performed in a
MJ Scientific thermal cycler at 50C for 30 min, followed by amplification by 30 cycles of 30 s at 94C, 30 s at 55 C,
1 min at 72C, and a final 10 min at 72 C extension time using the following human⁄ mouse-specific primers: HME1 CCAGATGTCACATGACAGTGGAAAGCACTA; HME2 CCGGGTTGGCATGACTCATATTAACCATGG; UXT 5¢-GACAAGGTATATGAGCAGCTG; UXT 3¢-TTG ATATTCATGGAGTCCTTG; Gapdh5 ACCACAGTCCA TGCCATCAC; Gapdh3 TCCACCACCCTGTTGCTGTA PCR products were resolved by agarose gel electrophoresis (NuSieve GTG agarose, FMC)
Sequencing
A Licor automated sequencer was used for sequence determ\ination using SequiTherm EXCELII (Cambio, Cambridge, UK) and appropriate IRD-800 labelled primers
Site-directed mutagenesis
The QuickChange XL system (Stratagene) was used accord-ing to the manufacturer’s instructions
Western blot analysis
Whole-cell extracts were prepared as described previously [14] Proteins were examined by SDS⁄ PAGE, transferred to HybondTM-ECL nitrocellulose, incubated with appropriate antibodies and visualized with an ECL western blotting detection system (Amersham Pharmacia Biotech, Piscata-way, NJ) Protein sizes were estimated by comparison with Full Range RainbowTM molecular mass markers (Amer-sham Pharmacia Biotech)