apart from global chromatin decondensation [ 47 ] , the mechanisms and molecules involved in nuclear reprogramming and in establishing pluripotency are not well understood .
oct4 is a gene that is normally expressed only in pluripotent , embryo - like cells .
it is not expressed in somatic cells but is activated upon nuclear transfer into oocytes and in embryonic stem ( es ) cells [ 2 , 8 ] .
when development and differentiation proceed [ 912 ] , or when retinoic acid is present , oct4 transcription is downregulated .
pluripotency proteins such as oct4 and nanog are transcription factors with essential roles in permitting the proliferation of es cells [ 1416 ] . even though several proteins , such as gcnf , sf1 , lrh1 , retinoic acid , and sall4 , are already known to regulate the oct4 promoter region in cultured cells or in later development , it is still unknown what is responsible for the activation of oct4 transcription , which correlates with cloning efficiency [ 21 , 22 ] .
xenopus oocytes provide abundant material and contain proteins that cause a large increase in the level of oct4 mrna .
the identification of such proteins is important for a better understanding of generating pluripotency and of the molecular events by which eggs or oocytes reprogram somatic - cell nuclei . to identify proteins that bind to the oct4 regulatory region ,
we have used radioactively labeled dna oligos corresponding to regions of the mouse oct4 promoter ( figures 1a and 1b ) .
these oligos were incubated in oocyte extract and dna - protein complexes analyzed on gels .
many of the oocyte - derived proteins showed sequence specificity for the oligos representing the oct4 regulatory region , as shown by the addition of increasing concentrations of unlabeled competitor dna ( figure  1c ) .
competition experiments with cpg methylated versus unmethylated oligos revealed that the proteins prefer to bind to the unmethylated oligos ( figure  1d ) .
to identify which proteins bind to the different oligos , we recovered the proteins from gels and identified them by mass spectrometry .
we have identified proteins already known to have a function in nuclear reprogramming and have thereby validated our procedure .
these proteins include nucleoplasmin [ 4 , 7 , 23 ] and the histone - binding protein n1/n2 .
we identified frgy2 , which is involved in disassembling somatic nucleoli in egg cytoplasm ; this disassembly process accompanies nuclear reprogramming .
retinoic acid induces retinoic - acid receptors in cultured cells , and an increase in both retinoic acid and its receptors causes differentiation of pluripotent cells [ 13 , 18 ] .
a retinoic - acid receptor has already been shown to bind to the oct4 regulatory region .
most intriguingly , we have identified tpt1 by using mass spectrometry with oligo sf1 ( data not shown ) ; tpt1 has not so far been implicated in regulating oct4 transcription .
it is widely expressed and conserved throughout vertebrates , and was first identified in a screen for genes involved in tumor reversion .
tpt1 is the gene that shows the strongest differential expression between tumor and tumor - reversed states in human leukemia and breast - cancer cells .
tpt1 is strongly expressed in tumor cells and is downregulated upon tumor reversal . moreover ,
when tpt1 is inhibited in tumor cells , suppression of the malignant phenotype is observed .
we repeated the gel shift , but this time in the presence of a tpt1 antibody ( figure  2a ) .
this resulted in a supershift , indicating that tpt1 in fact binds to the sf1  oligo and is not an artifact of the mass - spectrometry results .
in addition , tpt1 was also found to bind in vivo , as determined by chromatin immunoprecipitation ( chip ) analysis with xenopus oocytes ( figure  2b ) . to determine the developmental function of tpt1
, we carried out a test that depends on the downregulation of the tpt1 mrna .
we wanted to find out whether oct4 activation is inhibited in reduced abundance of tpt1 .
antisense dna oligos ( odn , oligo deoxynucleotides ) that successfully degrade endogenous tpt1 mrna in living oocytes were designed ( figures 2c and 2d ) .
antisense oligos were also prepared for the identified retinoic - acid receptor  ( rar ) , which serves as a control because of its known function in downregulating oct4 transcription   ( figures 2c and 2e ) .
after downregulation of both tpt1 and rar mrnas , the levels of other oocyte mrnas were unchanged ( figure  2f ) .
as a control , antisense oligos against gfp did not cause downregulation of any other genes tested ( figures 2c2f ) .
moreover , antisense oligos against xenopus tpt1 mrna resulted in specific downregulation of xenopus tpt1 protein but not of mouse tpt1 protein , which is not targeted by these antisense oligos ( figure  2 g ) . to reinforce our evidence for the importance of tpt1 for oct4 activation
, we have asked whether tpt1 is required for an increase in oct4 mrna in nuclear - transfer experiments .
forty hours after oocyte injection of antisense oligos , approximately 100 hela nuclei were injected into the germinal vesicles of oocytes , which were cultured for up to 24 hr . we took different time points and analyzed them by rt - pcr in order to observe the activation of oct4 ( figure  3a ) . because our method is very sensitive , we found that oct4 and nanog were expressed in nonpluripotent cells , such as human hela cells , but at a very low background level .
however , if a large number ( several thousand ) of nonpluripotent hela cells was analyzed , the signal was strong and was used as a positive control for the rt - pcr . the time course revealed a gradual increase of oct4 mrna ( figure  3c ) . in control samples that were only injected with hela nuclei and not antisense oligos , we observed a 100-fold increase in oct4 mrna within several hours of nuclear transfer ( yen - hsi kuo , personal communication ) .
we find that oocytes depleted of rar mrna activate human oct4 transcription earlier and more strongly than do oocytes with a normal endogenous level of rar ( figure  3c ) .
this increased level of oct4 transcription due to rar mrna downregulation agrees with previous findings that rar is an inhibitor of pluripotency and induces differentiation [ 13 , 18 ] and thus validates our functional test .
when antisense oligos against tpt1 were injected into oocytes , the opposite result was obtained .
we found that a reduction in the oocyte content of tpt1 greatly reduced oct4 transcription ( figure  3d ) .
this suggests that tpt1 is involved in oct4 transcriptional activation . to test the specificity of our antisense oligo against tpt1 mrna
, we have rescued xenopus tpt1 depletion by injecting mouse tpt1 mrna into oocytes previously injected with antisense oligos against tpt1 ( figure  3e ) .
importantly , mouse tpt1 mrna does not contain the sequence that is targeted by the xenopus antisense oligos . hence , mouse tpt1 mrna was not degraded and rescued the depletion of xenopus tpt1 mrna and , subsequently , oct4 expression ( figure  3e ) .
this result confirms that the downregulation of tpt1 mrna is specific . to determine more precisely how the level of tpt1 influences oct4 transcription , we injected different concentrations of mouse tpt1 mrna into oocyte cytoplasm .
an incubation period of 16 hr allowed for the mrna to be translated , and hela nuclei were then injected into the oocyte germinal vesicle .
after 8 hr of incubation , the oct4 signal was already enhanced in comparison to control oocytes ( figure  3f ) . as a result ,
this suggests that tpt1 might have an important role in regulating oct4 ; namely , it might ensure a level required for pluripotency and normal development .
we were interested in whether tpt1 is a transcription factor specific for oct4 or whether it is a more generally important component that might act on other pluripotency genes .
using the same up- and down - regulation procedure just described for oct4 , we have tested the transcriptional activation of the human pluripotency gene nanog by using nuclear transfer to oocytes ( figure  3 g ) [ 27 , 28 ] .
nanog seems to be an important pluripotency gene because its overexpression increases the efficiency of nuclear reprogramming by 200-fold in cell - fusion experiments .
overall , we found that  a reduction in the rar did not significantly affect nanog expression .
however , we had expected some effect on nanog expression because retinoic acid is known to downregulate pluripotency genes [ 13 , 18 ] .
one explanation for our finding is that nanog or nanog orthologs do not exist in amphibians ( ncbi blast ) . as a result
, some aspects of transcriptional activation upon nuclear transfer might not be conserved between mammals and amphibians .
however , we saw a reduction in nanog transcription after downregulation of tpt1 in oocytes ( figure  3 g ) . to test for binding of tpt1 to nanog , we carried out chip analysis in mouse es cells and found that tpt1 bound to oct4 , but not significantly to the immediate promoter of nanog ( figure  3b ) .
this suggests that tpt1 regulates oct4 by binding to its dna directly , whereas tpt1 might regulate nanog in an indirect way or by binding to a distant site .
we suggest that tpt1 might have a somewhat general role in establishing an embryonic pattern of gene expression because it controls transcription of oct4 and nanog . in support of this view
, we have also found that the transfection of tpt1 antisense rna into mouse es cells can reduce the transcription from the oct4 promoter ( figures 4a4c ) , suggesting a conserved mechanism between mammals and amphibians .
the molecular events occurring after nuclear transfer and the initiation of pluripotency are not well understood .
we describe here the first protein that has been discovered to be required for activating transcription of pluriptoency genes such as oct4 and nanog after nuclear transfer into oocytes . in the future
, this type of work might provide a generally applicable experimental design , which in combination with the advantages of xenopus oocytes , might lead to the identification of other proteins that activate pluripotency genes and thereby further elucidate mechanisms involved in nuclear reprogramming .
complementary single - stranded 30 bp oligos that represent dna fragments of the oct4 promoter   were annealed to double - stranded ( ds ) dna .
the phosphate binding buffer consisted of 20 mm phosphate ( ph  7.4 ) , 10% glycerol , 3 mm mgcl2 , 17 mm kcl , 2.5 m dtt , and 50  mm2 m nacl .
we used 8 l oocyte extract per sample and prepared it by mixing freshly obtained oocytes in presence of protease inhibitors . centrifuging the mixture overnight eliminated debris and lipids .
for the gel shift , 8 l oocyte extract , 2 g bsa , and 10 l phosphate binding buffer were mixed with approximately 0.1 pmol radioactively labeled oligos and run on a native gel . to confirm binding of tpt1 to any one oligo
, we added 1 l of tpt1 antibody ( monoclonal antibody anti - hrf / tctp , medical & biological laboratories ) to the sample before was running it on a gel .
the samples were  washed , concentrated , and analyzed with maldi - tof mass spectrometry .
 summarynuclear transfer to eggs or oocytes provides a potential route for cell - replacement therapies [ 1 ] because oocytes directly reprogram transplanted mammalian somatic - cell nuclei such that they have an embryo - like pattern of gene expression . 
 this includes a large increase in the mrna level of the stem - cell marker gene oct4[2 ] . 
 we have developed a novel procedure to identify new proteins that greatly increase the level of oct4 mrna upon nuclear transfer . 
 we have isolated xenopus oocyte proteins that bind to the regulatory region of the mouse oct4 gene and identified these by mass spectrometry . 
 the proteins include the retinoic - acid - receptor  , a known repressor of oct4 transcription , and tpt1 , a cancer - associated factor [ 3 ] . 
 the depletion of transcripts of retinoic - acid receptor  from oocytes increases oct4 and nanog transcription as expected , and depletion of tpt1 transcripts in oocytes reduces oct4 and nanog transcription in injected hela nuclei . 
 an elevation of tpt1 transcripts in oocytes results in an earlier activation of oct4 transcription . 
 therefore , we identify a novel role for tpt1 in activating pluripotency genes upon nuclear transfer . our results help to elucidate the mechanism by which somatic - cell nuclei are reprogrammed to have an embryo - like pattern of gene expression . 
Results and Discussion
Experimental Procedures