BACKGROUND
transcriptional intermediary factor tif1β and heterochromatin protein  <dig> profoundly impact the regulation of the structure and function of chromatin  <cit> .
the heterochromatin protein  <dig>  family of proteins  participates in gene silencing by forming heterochromatic structures  <cit> .
hp <dig> exhibits distinct nuclear localization patterns: hp1α nassociates with centromeres while hp1β and hp1γ are largely localized in distinct nuclear regions.
the nuclear arrangement of hp <dig> proteins and tif1β is differentiation pathway-specific, and appears to be more important than changes in the levels of these proteins, which are relatively stable during all of the induced differentiation processes  <cit> .
hp <dig> proteins comprise an n-terminal chromodomain, a c-terminal chromoshadow domain and a hinge domain  <cit> .
the chromodomain functions as a protein interaction domain, bringing together different proteins in multi-protein complexes and recruiting them to heterochromatin.
this domain binds to particular proteins which contain an hp <dig> box  and function at the transcriptional level  <cit> .
the chromoshadow domain mediates the formation of homodimer.
tif1β interacts with the chromodomain of dimeric hp1β via the hp <dig> box of tif1β in the hp1-interaction domain  <cit> .
the tif1β ntranscriptional repression activity depends on the interaction between tif1β and hp <dig>  <cit> .
this interaction is essential in the relocation of tif1β from euchromatin to heterochromatin that accompanies the differentiation of primitive endoderm-like cells  <cit> .
tif1β is proposed to function as a universal co-repressor protein for the krab zinc finger protein  superfamily of transcriptional repressors.
the recruitment of hp <dig> proteins by the krab-tif1β complex to specific loci within the genome via the formation of heterochromatin-like complexes may thus silence gene activity.
gene-specific repression may be a consequence of the formation of such complexes  <cit> .
it has been reported that tif1β directly interacts with the histone methyltransferase setdb <dig>  which methylates specifically histone h3/lys <dig> within euchromatin  <cit> .
depletion of endogenous levels of tif1β by sirna significantly inhibited the krab-mediated transcriptional repression of a chromatin template and inhibited cell cycle progression.
cell death may occur if tif1β is severely depleted by sirna knockdown .
similarly, the knock-down of cellular levels of hp <dig> proteins and setdb <dig> by sirna attenuated krab-tif1β repression.
the physiological targets and functions of tif1β remain unclear.
its interactions with chromatin modification factors such as hdac <dig>  setdb <dig> and hp <dig> correlate with its activities in regulating the chromatin structure and heterochromatin formation, resulting in epigenetic silencing of reporter genes  <cit> .
a tif1β-containing multiprotein complex regulates various protein activities or genes  <cit> .
the activity of tif1β may be regulated by posttranslational modifications.
for example, tif1β is rapidly phosphorylated by members of the phosphatidylinositol- <dig> kinase-like family of kinases following dna damage.
phosphorylated tif1β colocalizes with numerous damage response factors at dna lesions  <cit> .
recent genetic and proteomic studies in mice have shown that tif1β is a developmental regulatory protein performing cellular function that are critical to early embryonic development, and is a component of the interactive protein network for the pluripotency of embryonic stem cells  <cit> .
the binding of the tif1β corepressor to the retrovirus primer binding site is responsible for the epigenetic silencing of retrovirus transcription  <cit> .
therefore, the interaction between tif1β and hp <dig> or other transcription factors may account for the regulation of gene expression.
phosphorylation of both human and mouse tif1β/ser <dig> has been identified by nuclear phosphoprotein analysis of hela and wehi- <dig> cells  <cit> .
ser <dig> is located in the hp1-interacting domain of tif1β – close to the hp <dig> box .
the conservation of tif1β/ser <dig> phosphorylation in various cell lines from different species motivated this investigation of the functional significance of this modification.
the coil-coiled domain of tif1β binds to e2f <dig> and inhibits its activity  <cit> .
the induction of cyclin a, cdc <dig> and cdc25a genes depends on e2f  <cit> .
cyclin a is a cell cycle-regulating protein that participates in s-phase control and mitosis in mammalian somatic cells.
the promoter of cyclin a is repressed during the g <dig> phase of the cell cycle and is activated at s-phase entry  <cit> .
cdc <dig> permits the transition from g <dig> through s in conjunction with cyclin e  <cit> .
these e2f downstream genes are important to normal cell cycle progression  <cit> .
this report shows that tif1β participates in the regulation of cyclin a <dig>  cdc <dig> and cdc25a gene expression.
this regulation depends on the interaction between tif1β and hp1β, which is itself regulated by the phosphorylation state of tif1β/ser <dig>  the experimental results suggest that the tif1β/ser473-unphosphorylated form binds more strongly than the phosphorylated form to the promoters of cyclin a <dig>  cdc <dig>  and cdc25a genes.
the phosphorylation/de-phosphorylation of tif1β/ser <dig> may serve as a molecular switch regulating its interaction with hp1β and gene expression.
RESULTS
characterization of tif1β and phosphorylated tif1β/ser <dig> antibodies
western blotting of interphase hela cell extract was performed to characterize the specificity of monoclonal antibody to tif1β, clone 20a <dig>  a specific band of  <dig> kda was detected .
293t cell-expressed flag-tif1β was recognized either by 20a <dig> or monoclonal anti-flag antibody, m <dig> , demonstrating that monoclonal antibody 20a <dig> is specific to tif1β.
the specificity of rabbit anti-phospho-ser <dig> antibody was characterized by western blotting of interphase 293t cell extract with rabbit anti-tif1β/phospho-ser <dig> antibody  or 20a <dig> monoclonal antibody.
the phosphorylated tif1β/ser <dig> signal was diminished after calf intestine alkaline phosphatase  treatment .
ectopically-expressed flag-tif1β was immunoprecipitated from 293t cells with m <dig> beads and treated with cip to examine whether the signal was due to the phosphorylation of tif1β/s <dig>  20a <dig> and s <dig> antibodies both recognized flag-tif1β, but the signal recognized by the s <dig> antibody disappeared upon cip treatment .
unlike the flag-tif1β, mutants flag-tif1β/s473a and flag-tif1β/s473e were not detected by s <dig> antibody .
these data demonstrate that while 20a <dig> or s <dig> antibodies recognize the same protein, tif1β, the s <dig> antibody specifically recognizes phosphorylated tif1β/s <dig>
phosphorylation of tif1β/ser <dig> is dynamically regulated during cell cycle progression
although tif1β is known to be a general corepressor protein, few studies have suggested that tif1β has a critical function in cell cycle progression.
the phosphorylation of tif1β/ser <dig> was identified in hela and wehi cells at interphase  <cit> .
since ser <dig> is located near the hp1-binding motif, an intriguing question arises: what is the functional consequence, if any, of the phosphorylation of tif1β/ser473?
to elucidate the phosphorylation state of tif1β/ser <dig> during cell cycle progression, western blotting was performed with lysates from synchronized hela cells.
cells were synchronized to g1/s transition using double thymidine block.
phosphorylated tif1β/ser <dig> was almost undetectable at the g1/s boundary under the thymidine block  but the phosphorylation of tif1β/ser <dig> was maximal  <dig> hour post-release from the thymidine block, during early s phase .
the level of phosphorylated tif1β/ser <dig> decreased thereafter for  <dig> hours during the mid to late s-phase cell cycle progression.
the level of total tif1β remained relatively constant .
the elevated cyclin a and decreased cyclin b evidenced the g1/s to s cell cycle progression of thymidine release .
hela cells were synchronized to prometaphase by nocodazole treatment.
the level of phosphorylated tif1β/ser <dig> was determined from hela extracts collected from  <dig> to  <dig> hours after nocodazole were removed.
the phosphorylated tif1β/ser <dig> level was highest during mitosis  and decreased thereafter through g <dig> phase .
the lower panel of figure 2b shows the level of phosphorylated tif1β/ser <dig> in relation to that of total tif1β at each time point.
the m-phase marker cyclin b or phosphorylated histone h3s <dig> identify specific stages of mitotic release .
to further evaluate the phosphorylation state of tif1β/s <dig> from g <dig> to s phase, hela cells were serum starved for three days to arrest in g <dig> phase.
the level of phosphorylated tif1β/ser <dig> was almost non-detectable in g <dig> phase , reached a maximum  <dig> hours after serum was added and declined thereafter .
the gradual increase in the level of cyclin a or decrease in the level of cyclin b demonstrated the specific cell cycle stage from g <dig> to s .
this dynamic, biphasic  appearance of phosphorylated tif1β/ser <dig> suggests that tif1β may be involved in regulating the cell cycle by the switching on and off of ser <dig> phosphorylation.
various cell cycle regulated proteins may exhibit changed activity when phosphorylation modulates their stability.
the total protein level of tif1β remained approximately constant while the level of phosphorylated tif1β/ser <dig> fluctuated throughout the cell cycle.
to further demonstrate this dynamic fluctuation of phosphorylated tif1β/ser <dig> level, hela cells were immunostained using s <dig> antibody.
the level of phosphorylated tif1β/ser <dig> in the cells  <dig> hr after serum addition markedly exceeded that in the serum-starved cells .
the protein level and distribution of tif1β during g <dig> were the same as those of early s phase .
furthermore, in later experiments using over-expressed flag-tif1βs, similar nuclear distribution patterns of over-expressed wild-type flag-tif1β, flag-tif1β/s473a and flag-tif1β/s473e were observed .
hp1β staining also showed nuclear localization with over-expressed flag-tif1βs .
as a well known corepressor, tif1β regulates gene expression through direct binding to hp <dig> proteins in numerous transcriptional-repressed loci.
thus, the immunostaining data further indicate that tif1β might control its co-regulator function in the nuclear microenvironment via ser <dig> phosphorylation/dephosphorylation.
phosphorylation of tif1β/ser <dig> compromises interaction between hp1β and tif1β
the ser <dig> is located close to the pxvxl -containing hp <dig> box  .
to elucidate the role of tif1β in regulating gene expression, we examined whether its interaction with hp1β was affected by the phosphorylation of s <dig>  immunoprecipitation and western blotting experiments were performed using 293t cells that were co-transfected with flag-tif1β , flag-tif1β/s473a  or flag-tif1β/s473e  and ha-hp1β.
the interaction between flag-tif1β/s473a and hp1β was stronger than that between flag-tif1β/s473e or flag-tif1β and hp1β .
to demonstrate that the reduced phosphorylation level of tif1β/ser <dig> was related to its stronger interaction with hp1β, a pull-down assay was performed using purified gst-hp1β from e. coli and flag-tif1β, flag-tif1β/s473a or flag-tif1β/s473e from 293t cells.
gst-hp1β interacted with non-phosphorylated-mimetic flag-tif1β/s473a  <dig>  times more effectively than with phosphorylated-mimetic flag-tif1β/s473e .
the similarity of the level of interaction between wild-type flag-tif1β and gst-hp1β and that of flag-tif1β/s473a and gst-hp1β  may reflect a lower phosphorylation level of wild-type flag-tif1β here.
to verify that tif1β/ser <dig> phosphorylation compromises the interaction between tif1β and hp1β, m <dig> or ha antibody pull-down assays were conducted using in vitro translated ha-hp1β and flag-tif1β, flag-tif1β/s473a, or flag-tif1β/s473e.
the results of the m <dig> pull-down and the ha antibody pull-down assay indeed demonstrated that the interaction between ha-hp1β and flag-tif1β/s473a is much stronger than that between flag-tif1β/s473e and flag-tif1β .
taken together, these data demonstrate that the phosphorylation state of tif1β/ser <dig> influences the interaction between tif1β and hp1β both in vivo and in vitro.
when tif1β/ser <dig> is phosphorylated, its interaction with hp1β is compromised.
although the tif1β co-repressor function is known to be related to hp1β, few studies have addressed the specific gene targets of tif1β.
tif1β/s <dig> phosphorylation is up-regulated during the s phase in hela cells and the interaction between hp1β and tif1β is compromised when ser <dig> is phosphorylated.
these observations suggest that the phosphorylation of tif1β/ser <dig> may regulate gene expression by abolishing its interaction with hp1β.
tif1β regulates key genes expression during g <dig> to s-phase cell cycle progression
chromatin immunoprecipitation  experiments were used to study the possible involvement of ser473-phosphorylated/dephosphorylated tif1β in the expression of hela cell cycle-specific genes during the transition from g <dig> phase to s phase.
tif1β/phospho-ser <dig> was almost undetectable in the g <dig> phase, and climbed thereafter to reach maximum level in early s phase.
as cyclin a <dig> is known to be expressed in early s phase but not in g <dig> phase, a reasonable question arises: is the tif1β/ser <dig> phosphorylation correlated with cyclin a2's expression?
pcr primers encompassing the e2f site were used to detect dna fragments corresponding to the promoter region of cyclin a <dig>  a much higher level of tif1β was associated with the cyclin a <dig> promoter at the g1/s boundary than in early s phase .
these results are closely correlated with the rise in phosphorylated tif1β/ser <dig> in the early s phase, but not at the g1/s boundary .
histone h <dig> k9dime was associated with the silencing of gene expression.
the chip results demonstrated that the level of h3k9dime correlated strongly with that of tif1β.
however, histone h3k4dime is known to associate with actively transcribed genes.
its association with the promoter region of cyclin a <dig> was inversely correlated with that of tif1β.
quantitative chip results comparing the cyclin a <dig> level in g1/s and s phase are presented under the panel of pcr results obtained using each probe.
as a control, tif1β was not associated with the promoter of cyclin e .
tif1β/phospho-ser <dig> was also elevated  <dig> hours after serum was added .
chip was thus used to measure the level of tif1β bound to the cyclin a <dig> promoter in g1-phase hela cells that had been starved of serum for three days or s-phase hela cells that had been released from serum starvation.
more tif1β in the g <dig> phase cells was associated with the promoter region of cyclin a <dig> .
this finding is closely correlated with the elevated level of phosphorylated tif1β/ser <dig> in the s phase but not in the g <dig> phase .
quantitative chip results, comparing cyclin a <dig> levels in the g <dig> and s phase, are presented beneath the pcr results obtained using each probe.
taken together, these results indicate that the association of tif1β with the promoter of cyclin a <dig> is correlated with silencing while dissociation is correlated with derepression of the promoter.
immunostaining showed that over-expression of flag-tif1β, flag-tif1β/s473a, and flag-tif1β/s473e did not alter their nuclear localization .
a flow cytometric analysis was performed to test further the effects of over-expressed flag-tif1β, flag-tif1β/s473a and flag-tif1β/s473e on cell cycle progression.
over-expression of the flag-tif1β/s473a mutant caused an accumulation of cells stalled at g2/m in 293t cells , while the profiles of cell cycle progression were similar in 293t cells over-expressing flag-tif1β and flag-tif1β/s437e, suggesting that the phosphorylation state of tif1β/ser <dig> affects cell cycle progression.
quantitative real-time pcr analysis of 293t cells revealed half the amount of cyclin a <dig> mrna in the flag-tif1β/s473a-overexpressing cells compared to the falg-tif1β or falg-tif1β/s473e-expressing cells .
the phenotype of the g2/m stalls and the reduction of the cyclin a <dig> mrna level in tif1β/s473a over-expressing 293t cells are consistent with observations made in a gfp-cyclin a <dig> sirna knockdown experiment published by kenrick et al.
<cit> .
taken together, these results suggest that disruption of tif1β/ser <dig> phosphorylation may influence cell cycle-regulated gene expression, and that cyclin a <dig> may be one of the tif1β indirect target genes.
these data also demonstrate that the ser <dig> phosphorylation/dephosphorylation status of tif1β may regulate cell cycle progression.
to further examine the association of unphosphorylated tif1β/ser <dig> with other cell cycle-regulated genes, such as cdc <dig> and cdc25a, quantitative chip experiments were conducted with hek293t cells that had been transfected with ha-hp1β and flag-tif1β, flag-tif1β/ser473a, and flag-tif1β/ser473e.
when chip was performed with ha monoclonal antibody, the association of hp1β with the promoters of cdc <dig> or cdc25a in flag-tif1β/ser473a over-expressing cells was stronger than in flag-tif1β/ser473e-over-expressing cells .
when chip was performed using 20a <dig> , no obvious difference between flag-tif1β/ser473a and flag-tif1β/ser473e was observed.
collectively, these results reveal that over-expressed hp1β and tif1β/ser473a may form a stronger complex and preferentially associate with the promoter regions of cdc <dig> and cdc25a genes more than over-expressed hp1β and tif1β/ser473e in interphase hek293t cells.
phosphorylation of tif1β ser <dig> in s phase is mediated by pkc pathway
tif1β/ser <dig> phosphorylation is up-regulated in s phase.
to identify which kinase are involved in the phosphorylation of tif1β/ser <dig> in the s phase, we used western blotting of cell extracts prepared from cells that had been treated with a panel of kinase inhibitors.
among the inhibitors tested, only the pkc inhibitor  exhibited significant inhibition, while the ck <dig> inhibitor had a moderate effect on the phosphorylation of ser <dig> .
an inhibitory peptide  that contained tif1β/s471a/s473a also blocked tif1β/ser <dig> phosphorylation .
neither camk-ii inhibitor nor staurosporine had any effect .
to confirm that the phosphorylation of tif1β/ser <dig> proceeds along the pkc pathway, the effect of 12-o-tetradecanoylphorbol-13-acetate  on tif1β/ser <dig> phosphorylation in interphase hela cells was tested.
tpa treatment induced dramatic phosphorylation of tif1β/ser <dig> within  <dig> hour .
to further examine whether subtype-pkcδ is involved in the phosphorylation of tif1β/ser <dig>  m2-immunoprecipitation and western blotting with s <dig> antibody were performed using cell extracts that were prepared from flag-tif1β and ha-pkcδ-cotransfected 293t cells.
the phosphorylation level of tif1β/ser <dig> was double that of the control .
immunostaining also revealed an increase of the tif1β/phospho-ser <dig> signal in ha-pkcδ-transfected hela cells .
although the majority of over-expressed pkcδ was located in the cytoplasm, a minor portion appeared in the nucleus .
collectively, these results indicate that tif1β/ser <dig> phosphorylation in s phase could be mediated by the pkcδ pathway, and that tif1β/ser <dig> phosphorylation correlated with the expression of target genes by weakening tif1β/hp1β interaction .
level of phosphorylated tif1β/ser <dig> is reduced in megakaryocytic differentiated k <dig> cells
tif1β/ser <dig> phosphorylation fluctuates during cell cycle progression.
k <dig> cells were used as an inducible cell differentiation system to profile the level of phosphorylated tif1β/ser <dig> in both proliferating and differentiated cells.
k <dig> is an erythroleukemic cell line that can undergo further differentiation in both megakaryocytic and erythroid lineages, depending on the stimulus  <cit> .
it is a well-characterized system in which tpa stimulates the megakaryocytic development of k <dig>  western blotting was performed on extracts prepared from proliferating or tpa-induced differentiated k <dig> cells.
the differentiated k <dig> cells exhibited megakaryocytic morphology , as demonstrated by phase contrast images .
although the total tif1β protein level in differentiated k <dig> cells was lower than that in proliferating cells , the level of phosphorylated tif1β/ser <dig> was halved in megakaryocytic differentiated k <dig>  consistent with the observation that tif1β promotes cell cycle progression by the up-regulation of its ser <dig> phosphorylation, the amount of tif1β/ser <dig> phosphorylation may be utilized as a proliferation marker in cancer cells.
discussion
this investigation demonstrates that the phosphorylation of tif1β/ser <dig> by kinase  may act as a molecular switch in the temporal regulation of gene expression.
tif1β/ser <dig> is located close to the hp <dig> box, the pxvxl motif, which is responsible for the interaction between tif1β and hp <dig>  and its phosphorylation negatively affects this interaction.
the dynamic phosphorylation of tif1β/ser <dig> suggests that this event may regulate the functions of tif1β in cell cycle progression.
the central question then is: how may the phosphorylation of tif1β/ser <dig> regulate tif1β-mediated gene expression?
we addressed this question by investigating the potential regulatory functions of tif1β on key genes for cell cycle pregression and the effects of over-expressing phosphorylation-deficient or phosphomimetic mutants of tif1β/ser <dig> on cell cycle progression.
tif1β is proposed to function as a universal corepressor protein for the krab zinc finger protein  superfamily of transcriptional repressors  <cit> .
the recruitment of hp <dig> proteins by the krab-tif1β complex to specific loci within the genome through the formation of heterochromatin-like complexes may silence gene activity.
the phosphorylation of tif1β/s <dig> compromises its interaction with hp1β in a manner that is related to cell cycle-regulated gene expression
the disruption of the interaction between hp1β and tif1β by the phosphorylation of tif1β/ser <dig>  suggests that this phosphorylation/dephosphorylation of tif1β/ser <dig> may be the means of regulation of tif1β- and hp1β-mediated gene silencing.
the results of chip experiments suggest that the majority of tif1β associated with the promoters of cyclin a <dig> in g <dig> phase cells is likely to be unphosphorylated tif1β/ser <dig>  this conclusion is supported by two lines of evidence:  very low levels of phosphorylated tif1β/ser <dig> were observed in g <dig> cells, and  overexpressed flag-tif1β/s473a bound to the promoters of cdc <dig> and cdc25a better than flag-tif1β/s473e.
when cells were released into the s phase, the association of unphosphorylated tif1β/ser <dig> with these promoters decreased, accompanying an increased level of phosphorylated tif1β/ser <dig>  the dynamics of tif1β/ser <dig> phosphorylation and tif1β-binding to the cyclin a <dig> promoter  indicate that un-phosphorylated tif1β/ser <dig> is responsible for silencing the cyclin a <dig> gene in the g <dig> phase .
the observation that hp1β interacted strongly with un-phosphorylated tif1β/ser <dig> is consistent with the chip results concerning the over-expressed recombinant variants, where over-expressed flag-tif1β/s473a associated better with the promoter region of cdc <dig> or cdc25a than that did flag-tif1β/s473e .
the treatment of cells with cyclin a <dig> sirna led to an accumulation of cells in prophase and mitosis to a degree similar to that observed for cyclin b <dig>  consistent with the requirement of cyclin a for g1/s and g2/m transitions  <cit> .
interestingly, the chip results  and the effects of over-expressed flag-tif1β/ser473a on cell cycle progression  are consistent with published results for the sirna knockdown of cyclin a <dig>  <cit> .
hp1β recruitment to e2f-binding element of cdc <dig> and cdc25a promoters was affected by the phosphorylation state of tif1β/ser <dig>  the level of hp1β recruitment to cdc <dig> or cdc25a promoter was increased when wild-type flag-tif1β or flag-tif1β/s473a were ectopically expressed.
this association was compromised by the phosphomimetic mutant, s473e, which suggests that hp1β recruitment is negatively regulated by phosphorylation of tif1β/ser <dig>  likewise, ectopically expressed hp1β resulted in elevated recruitment of wild-type flag-tif1β or flag-tif1β/s473a.
these observations provide a clue that the recruitment of tif1β and hp1β could work in a positive feedback manner.
the above results demonstrate that the dynamic interconversion between unphosphorylated and phosphorylated forms of tif1β/ser <dig> may be crucial in the regulation of gene expression during cell cycle progression.
despite extensive efforts to perform chip with rabbit anti-phosphorylated tif1β/ser <dig> antibody, no significant result was obtained.
a likely explanation of this failure is that phosphorylated tif1β/ser <dig> is not associated with the promoter of cyclin a <dig> in the g <dig> phase or, alternatively, the epitope in the multiprotein complex is masked from antibody access.
the way in which tif1β disassociates from the hp1-containing heterochromatin is unclear.
however, the findings herein provide a molecular explanation, which involves pkc-mediated phosphorylation at tif1β/ser <dig> .
phosphorylated tif1β/s <dig> level may serve as a proliferation marker
tif1β transcriptional repression activity depends on the interaction between tif1β and hp1β  <cit> .
this interaction is essential to the relocation of tif1β from euchromatin to heterochromatin that accompanies the differentiation of primitive endoderm-like cells  <cit> .
tif1β is known to interact differentially with hp1β and hp1γ in differentiated and non-differentiated cells  <cit> .
in non-differentiated cells, tif1β/hp <dig> interaction occurs only in euchromatin and selectively involves hp1β and hp1γ, but not hp1α.
in differentiated cells, on the other hand, tif1β selectively associates with hp1β in heterochromatin, while tif1β and hp1γ interaction occurs only in euchromatin.
these conclusions agree with the reduced level of phosphorylated tif1β/ser <dig> seen here in differentiated k <dig> cells .
the results herein also revealed that un-phosphorylated tif1β/ser <dig> interacts more strongly with hp1β than its phosphorylated counterpart .
these results further suggest that the phosphorylation of tif1β/ser <dig> regulates the differential interaction between tif1β and hp1β.
what may be the functional consequences of the phosphorylation of tif1β/ser <dig> and its consequent dissociation from hp1s must be addressed.
tif1β interacts with e2f  <cit> , trip-br and cbp/p <dig>  and potentiates the co-activation of e2f-1/dp- <dig> by trip-br protein  <cit> .
phosphorylated tif1β/ser <dig> may thus be involved in this tripartite functional interaction.
this suggestion is supported by our findings that the induction of cyclin a <dig> is accompanied by an increased level of phosphorylated tif1β/ser <dig> and reduced binding of tif1β to the promoter.
a most provocative question that remains to be answered is whether the ser473-phosphorylated tif1β may interact preferentially with transcription factors and serve as a coactivator.
the results also demonstrated that the level of phosphorylated tif1β/ser <dig> peaks at early s- and m-phases.
two phosphorylation sites other than ser <dig>  ser <dig> and ser <dig>  were also identified in the course of this investigation.
both sites are located in the bromodomain of tif1β.
the phosphorylation-deficient or phosphomimetic mutants of ser <dig> did not influence the phosphorylation of ser <dig> , suggesting that these phosphorylations may be independent events during mitosis.
other phosphorylation sites important for regulating the function of tif1β have also been identified  <cit> .
the functional consequences of ser <dig> and ser <dig> phosphorylation remain to be investigated.
pkc mediated tif1β/s <dig> phosphorylation may be involved in cell cycle progression
numerous examples have demonstrated that pkcδ is centrally involved in cell proliferation and differentiation.
the activation of pkcδ uniquely mediates insulin-induced proliferation: pkcδ is activated by insulin and interacts with insulin receptor and irs  <cit> .
insulin-activated pkcδ interacts with 3-phosphoinositide-dependent protein kinase to regulate protein kinase b  <cit>  and is responsible for stat <dig> activation and keratinocyte proliferation  <cit> .
although the over-expressed pkcδ is mainly located in the cytoplasm , it has been shown here  and by kajimoto et al.
to partially localize to the nucleus  <cit> .
the observation herein that the serum-stimulated phosphorylation of tif1β/ser <dig> correlates strongly with g1/s progression and cyclin a <dig> expression  uncovers a novel mechanism of pkcδ-mediated g <dig> to s phase cell cycle progression.
phosphorylation of tif1β/ser <dig>  gene activation and stem cell proliferation
the dynamic phosphorylation of tif1β/ser <dig> during cell proliferation and differentiation  suggests that phosphorylation/dephosphorylation is crucial for regulating the transcriptional activity of tif1β.
during the differentiation of embryonic carcinoma cells, the intracellular distribution of tif1β changes from diffuse nuclear staining to discrete foci and colocalizes with heterochromatin  <cit> .
the steady-state level of tif1β is also decreased.
the reduced levels of phosphorylated tif1β/ser <dig> as well as the lower steady-state tif1β levels in differentiated cells suggest that tif1β may be mainly localized to heterochromatin in differentiated cells.
in embryonic stem cells, tif1β is present in complexes with various pluripotent markers, including rex- <dig>  dax- <dig> and nanog  <cit> .
since phosphorylated tif1β/ser <dig> seems to be preferentially associated with cell proliferation, it is important to determine whether it resides in these complexes.
in fact, the level of phosphorylated tif1β/ser <dig> may serve as a proliferation marker.
the epigenetic silencing of retrovirus transcription is caused by the binding of a tif1β corepressor complex to retrovirus primer binding site  <cit> .
the likely recruitment of tif1β by a dna-binding krab-box containing zinc finger protein for pbs-mediated silencing should be addressed, and the question of whether the regulation of the phosphorylation of tif1β/ser <dig> or the formation of tif1β corepressor complex is participating in retrovirus transcription should also be investigated.
CONCLUSIONS
although the tif1β co-repressor function is known to be related to hp1β, few studies have addressed the specific gene targets of tif1β.
we have found that cell cycle progression is regulated by tif1β.
the key cell cycle regulatory genes, cyclin a <dig>  cdc <dig> and cdc25a are targeted by tif1β, and phosphorylation of tif1β/ser <dig> is associated with the activation of these genes.
tif1β/s <dig> phosphorylation is up-regulated during the s phase in hela cells and the interaction between hp1β and tif1β is compromised when ser <dig> is phosphorylated.
these observations suggest that the phosphorylation of tif1β/ser <dig> may regulate gene expression by abolishing its interaction with hp1β.
thus, phosphorylation of tif1β/ser <dig> plays a crucial role in epigenetic regulation of gene expression.

 transcriptional intermediary factor tif1β and heterochromatin protein profoundly impact the regulation of the structure and function of chromatin. the heterochromatin protein family of proteins participates in gene silencing by forming heterochromatic structures. hp exhibits distinct nuclear localization patterns: hp1α nassociates with centromeres while hp1β and hp1γ are largely localized in distinct nuclear regions. the nuclear arrangement of hp proteins and tif1β is differentiation pathway-specific, and appears to be more important than changes in the levels of these proteins, which are relatively stable during all of the induced differentiation processes. hp proteins comprise an n-terminal chromodomain, a c-terminal chromoshadow domain and a hinge domain. the chromodomain functions as a protein interaction domain, bringing together different proteins in multi-protein complexes and recruiting them to heterochromatin. this domain binds to particular proteins which contain an hp box and function at the transcriptional level. the chromoshadow domain mediates the formation of homodimer. tif1β interacts with the chromodomain of dimeric hp1β via the hp box of tif1β in the hp1-interaction domain. the tif1β ntranscriptional repression activity depends on the interaction between tif1β and hp. this interaction is essential in the relocation of tif1β from euchromatin to heterochromatin that accompanies the differentiation of primitive endoderm-like cells. tif1β is proposed to function as a universal co-repressor protein for the krab zinc finger protein superfamily of transcriptional repressors. the recruitment of hp proteins by the krab-tif1β complex to specific loci within the genome via the formation of heterochromatin-like complexes may thus silence gene activity. gene-specific repression may be a consequence of the formation of such complexes. it has been reported that tif1β directly interacts with the histone methyltransferase setdb which methylates specifically histone h3/lys within euchromatin. depletion of endogenous levels of tif1β by sirna significantly inhibited the krab-mediated transcriptional repression of a chromatin template and inhibited cell cycle progression. cell death may occur if tif1β is severely depleted by sirna knockdown. similarly, the knock-down of cellular levels of hp proteins and setdb by sirna attenuated krab-tif1β repression. the physiological targets and functions of tif1β remain unclear. its interactions with chromatin modification factors such as hdac setdb and hp correlate with its activities in regulating the chromatin structure and heterochromatin formation, resulting in epigenetic silencing of reporter genes. a tif1β-containing multiprotein complex regulates various protein activities or genes. the activity of tif1β may be regulated by posttranslational modifications. for example, tif1β is rapidly phosphorylated by members of the phosphatidylinositol- kinase-like family of kinases following dna damage. phosphorylated tif1β colocalizes with numerous damage response factors at dna lesions. recent genetic and proteomic studies in mice have shown that tif1β is a developmental regulatory protein performing cellular function that are critical to early embryonic development, and is a component of the interactive protein network for the pluripotency of embryonic stem cells. the binding of the tif1β corepressor to the retrovirus primer binding site is responsible for the epigenetic silencing of retrovirus transcription. therefore, the interaction between tif1β and hp or other transcription factors may account for the regulation of gene expression. phosphorylation of both human and mouse tif1β/ser has been identified by nuclear phosphoprotein analysis of hela and wehi- cells. ser is located in the hp1-interacting domain of tif1β – close to the hp box. the conservation of tif1β/ser phosphorylation in various cell lines from different species motivated this investigation of the functional significance of this modification. the coil-coiled domain of tif1β binds to e2f and inhibits its activity. the induction of cyclin a, cdc and cdc25a genes depends on e2f. cyclin a is a cell cycle-regulating protein that participates in s-phase control and mitosis in mammalian somatic cells. the promoter of cyclin a is repressed during the g phase of the cell cycle and is activated at s-phase entry. cdc permits the transition from g through s in conjunction with cyclin e. these e2f downstream genes are important to normal cell cycle progression. this report shows that tif1β participates in the regulation of cyclin a cdc and cdc25a gene expression. this regulation depends on the interaction between tif1β and hp1β, which is itself regulated by the phosphorylation state of tif1β/ser the experimental results suggest that the tif1β/ser473-unphosphorylated form binds more strongly than the phosphorylated form to the promoters of cyclin a cdc and cdc25a genes. the phosphorylation/de-phosphorylation of tif1β/ser may serve as a molecular switch regulating its interaction with hp1β and gene expression.  characterization of tif1β and phosphorylated tif1β/ser antibodies western blotting of interphase hela cell extract was performed to characterize the specificity of monoclonal antibody to tif1β, clone 20a a specific band of kda was detected. 293t cell-expressed flag-tif1β was recognized either by 20a or monoclonal anti-flag antibody, m, demonstrating that monoclonal antibody 20a is specific to tif1β. the specificity of rabbit anti-phospho-ser antibody was characterized by western blotting of interphase 293t cell extract with rabbit anti-tif1β/phospho-ser antibody or 20a monoclonal antibody. the phosphorylated tif1β/ser signal was diminished after calf intestine alkaline phosphatase treatment. ectopically-expressed flag-tif1β was immunoprecipitated from 293t cells with m beads and treated with cip to examine whether the signal was due to the phosphorylation of tif1β/s 20a and s antibodies both recognized flag-tif1β, but the signal recognized by the s antibody disappeared upon cip treatment. unlike the flag-tif1β, mutants flag-tif1β/s473a and flag-tif1β/s473e were not detected by s antibody. these data demonstrate that while 20a or s antibodies recognize the same protein, tif1β, the s antibody specifically recognizes phosphorylated tif1β/s phosphorylation of tif1β/ser is dynamically regulated during cell cycle progression although tif1β is known to be a general corepressor protein, few studies have suggested that tif1β has a critical function in cell cycle progression. the phosphorylation of tif1β/ser was identified in hela and wehi cells at interphase. since ser is located near the hp1-binding motif, an intriguing question arises: what is the functional consequence, if any, of the phosphorylation of tif1β/ser473? to elucidate the phosphorylation state of tif1β/ser during cell cycle progression, western blotting was performed with lysates from synchronized hela cells. cells were synchronized to g1/s transition using double thymidine block. phosphorylated tif1β/ser was almost undetectable at the g1/s boundary under the thymidine block but the phosphorylation of tif1β/ser was maximal hour post-release from the thymidine block, during early s phase. the level of phosphorylated tif1β/ser decreased thereafter for hours during the mid to late s-phase cell cycle progression. the level of total tif1β remained relatively constant. the elevated cyclin a and decreased cyclin b evidenced the g1/s to s cell cycle progression of thymidine release. hela cells were synchronized to prometaphase by nocodazole treatment. the level of phosphorylated tif1β/ser was determined from hela extracts collected from to hours after nocodazole were removed. the phosphorylated tif1β/ser level was highest during mitosis and decreased thereafter through g phase. the lower panel of figure 2b shows the level of phosphorylated tif1β/ser in relation to that of total tif1β at each time point. the m-phase marker cyclin b or phosphorylated histone h3s identify specific stages of mitotic release. to further evaluate the phosphorylation state of tif1β/s from g to s phase, hela cells were serum starved for three days to arrest in g phase. the level of phosphorylated tif1β/ser was almost non-detectable in g phase, reached a maximum hours after serum was added and declined thereafter. the gradual increase in the level of cyclin a or decrease in the level of cyclin b demonstrated the specific cell cycle stage from g to s. this dynamic, biphasic appearance of phosphorylated tif1β/ser suggests that tif1β may be involved in regulating the cell cycle by the switching on and off of ser phosphorylation. various cell cycle regulated proteins may exhibit changed activity when phosphorylation modulates their stability. the total protein level of tif1β remained approximately constant while the level of phosphorylated tif1β/ser fluctuated throughout the cell cycle. to further demonstrate this dynamic fluctuation of phosphorylated tif1β/ser level, hela cells were immunostained using s antibody. the level of phosphorylated tif1β/ser in the cells hr after serum addition markedly exceeded that in the serum-starved cells. the protein level and distribution of tif1β during g were the same as those of early s phase. furthermore, in later experiments using over-expressed flag-tif1βs, similar nuclear distribution patterns of over-expressed wild-type flag-tif1β, flag-tif1β/s473a and flag-tif1β/s473e were observed. hp1β staining also showed nuclear localization with over-expressed flag-tif1βs. as a well known corepressor, tif1β regulates gene expression through direct binding to hp proteins in numerous transcriptional-repressed loci. thus, the immunostaining data further indicate that tif1β might control its co-regulator function in the nuclear microenvironment via ser phosphorylation/dephosphorylation. phosphorylation of tif1β/ser compromises interaction between hp1β and tif1β the ser is located close to the pxvxl -containing hp box. to elucidate the role of tif1β in regulating gene expression, we examined whether its interaction with hp1β was affected by the phosphorylation of s immunoprecipitation and western blotting experiments were performed using 293t cells that were co-transfected with flag-tif1β, flag-tif1β/s473a or flag-tif1β/s473e and ha-hp1β. the interaction between flag-tif1β/s473a and hp1β was stronger than that between flag-tif1β/s473e or flag-tif1β and hp1β. to demonstrate that the reduced phosphorylation level of tif1β/ser was related to its stronger interaction with hp1β, a pull-down assay was performed using purified gst-hp1β from e. coli and flag-tif1β, flag-tif1β/s473a or flag-tif1β/s473e from 293t cells. gst-hp1β interacted with non-phosphorylated-mimetic flag-tif1β/s473a times more effectively than with phosphorylated-mimetic flag-tif1β/s473e. the similarity of the level of interaction between wild-type flag-tif1β and gst-hp1β and that of flag-tif1β/s473a and gst-hp1β may reflect a lower phosphorylation level of wild-type flag-tif1β here. to verify that tif1β/ser phosphorylation compromises the interaction between tif1β and hp1β, m or ha antibody pull-down assays were conducted using in vitro translated ha-hp1β and flag-tif1β, flag-tif1β/s473a, or flag-tif1β/s473e. the results of the m pull-down and the ha antibody pull-down assay indeed demonstrated that the interaction between ha-hp1β and flag-tif1β/s473a is much stronger than that between flag-tif1β/s473e and flag-tif1β. taken together, these data demonstrate that the phosphorylation state of tif1β/ser influences the interaction between tif1β and hp1β both in vivo and in vitro. when tif1β/ser is phosphorylated, its interaction with hp1β is compromised. although the tif1β co-repressor function is known to be related to hp1β, few studies have addressed the specific gene targets of tif1β. tif1β/s phosphorylation is up-regulated during the s phase in hela cells and the interaction between hp1β and tif1β is compromised when ser is phosphorylated. these observations suggest that the phosphorylation of tif1β/ser may regulate gene expression by abolishing its interaction with hp1β. tif1β regulates key genes expression during g to s-phase cell cycle progression chromatin immunoprecipitation experiments were used to study the possible involvement of ser473-phosphorylated/dephosphorylated tif1β in the expression of hela cell cycle-specific genes during the transition from g phase to s phase. tif1β/phospho-ser was almost undetectable in the g phase, and climbed thereafter to reach maximum level in early s phase. as cyclin a is known to be expressed in early s phase but not in g phase, a reasonable question arises: is the tif1β/ser phosphorylation correlated with cyclin a2's expression? pcr primers encompassing the e2f site were used to detect dna fragments corresponding to the promoter region of cyclin a a much higher level of tif1β was associated with the cyclin a promoter at the g1/s boundary than in early s phase. these results are closely correlated with the rise in phosphorylated tif1β/ser in the early s phase, but not at the g1/s boundary. histone h k9dime was associated with the silencing of gene expression. the chip results demonstrated that the level of h3k9dime correlated strongly with that of tif1β. however, histone h3k4dime is known to associate with actively transcribed genes. its association with the promoter region of cyclin a was inversely correlated with that of tif1β. quantitative chip results comparing the cyclin a level in g1/s and s phase are presented under the panel of pcr results obtained using each probe. as a control, tif1β was not associated with the promoter of cyclin e. tif1β/phospho-ser was also elevated hours after serum was added. chip was thus used to measure the level of tif1β bound to the cyclin a promoter in g1-phase hela cells that had been starved of serum for three days or s-phase hela cells that had been released from serum starvation. more tif1β in the g phase cells was associated with the promoter region of cyclin a. this finding is closely correlated with the elevated level of phosphorylated tif1β/ser in the s phase but not in the g phase. quantitative chip results, comparing cyclin a levels in the g and s phase, are presented beneath the pcr results obtained using each probe. taken together, these results indicate that the association of tif1β with the promoter of cyclin a is correlated with silencing while dissociation is correlated with derepression of the promoter. immunostaining showed that over-expression of flag-tif1β, flag-tif1β/s473a, and flag-tif1β/s473e did not alter their nuclear localization. a flow cytometric analysis was performed to test further the effects of over-expressed flag-tif1β, flag-tif1β/s473a and flag-tif1β/s473e on cell cycle progression. over-expression of the flag-tif1β/s473a mutant caused an accumulation of cells stalled at g2/m in 293t cells, while the profiles of cell cycle progression were similar in 293t cells over-expressing flag-tif1β and flag-tif1β/s437e, suggesting that the phosphorylation state of tif1β/ser affects cell cycle progression. quantitative real-time pcr analysis of 293t cells revealed half the amount of cyclin a mrna in the flag-tif1β/s473a-overexpressing cells compared to the falg-tif1β or falg-tif1β/s473e-expressing cells. the phenotype of the g2/m stalls and the reduction of the cyclin a mrna level in tif1β/s473a over-expressing 293t cells are consistent with observations made in a gfp-cyclin a sirna knockdown experiment published by kenrick et al.. taken together, these results suggest that disruption of tif1β/ser phosphorylation may influence cell cycle-regulated gene expression, and that cyclin a may be one of the tif1β indirect target genes. these data also demonstrate that the ser phosphorylation/dephosphorylation status of tif1β may regulate cell cycle progression. to further examine the association of unphosphorylated tif1β/ser with other cell cycle-regulated genes, such as cdc and cdc25a, quantitative chip experiments were conducted with hek293t cells that had been transfected with ha-hp1β and flag-tif1β, flag-tif1β/ser473a, and flag-tif1β/ser473e. when chip was performed with ha monoclonal antibody, the association of hp1β with the promoters of cdc or cdc25a in flag-tif1β/ser473a over-expressing cells was stronger than in flag-tif1β/ser473e-over-expressing cells. when chip was performed using 20a, no obvious difference between flag-tif1β/ser473a and flag-tif1β/ser473e was observed. collectively, these results reveal that over-expressed hp1β and tif1β/ser473a may form a stronger complex and preferentially associate with the promoter regions of cdc and cdc25a genes more than over-expressed hp1β and tif1β/ser473e in interphase hek293t cells. phosphorylation of tif1β ser in s phase is mediated by pkc pathway tif1β/ser phosphorylation is up-regulated in s phase. to identify which kinase are involved in the phosphorylation of tif1β/ser in the s phase, we used western blotting of cell extracts prepared from cells that had been treated with a panel of kinase inhibitors. among the inhibitors tested, only the pkc inhibitor exhibited significant inhibition, while the ck inhibitor had a moderate effect on the phosphorylation of ser. an inhibitory peptide that contained tif1β/s471a/s473a also blocked tif1β/ser phosphorylation. neither camk-ii inhibitor nor staurosporine had any effect. to confirm that the phosphorylation of tif1β/ser proceeds along the pkc pathway, the effect of 12-o-tetradecanoylphorbol-13-acetate on tif1β/ser phosphorylation in interphase hela cells was tested. tpa treatment induced dramatic phosphorylation of tif1β/ser within hour. to further examine whether subtype-pkcδ is involved in the phosphorylation of tif1β/ser m2-immunoprecipitation and western blotting with s antibody were performed using cell extracts that were prepared from flag-tif1β and ha-pkcδ-cotransfected 293t cells. the phosphorylation level of tif1β/ser was double that of the control. immunostaining also revealed an increase of the tif1β/phospho-ser signal in ha-pkcδ-transfected hela cells. although the majority of over-expressed pkcδ was located in the cytoplasm, a minor portion appeared in the nucleus. collectively, these results indicate that tif1β/ser phosphorylation in s phase could be mediated by the pkcδ pathway, and that tif1β/ser phosphorylation correlated with the expression of target genes by weakening tif1β/hp1β interaction. level of phosphorylated tif1β/ser is reduced in megakaryocytic differentiated k cells tif1β/ser phosphorylation fluctuates during cell cycle progression. k cells were used as an inducible cell differentiation system to profile the level of phosphorylated tif1β/ser in both proliferating and differentiated cells. k is an erythroleukemic cell line that can undergo further differentiation in both megakaryocytic and erythroid lineages, depending on the stimulus. it is a well-characterized system in which tpa stimulates the megakaryocytic development of k western blotting was performed on extracts prepared from proliferating or tpa-induced differentiated k cells. the differentiated k cells exhibited megakaryocytic morphology, as demonstrated by phase contrast images. although the total tif1β protein level in differentiated k cells was lower than that in proliferating cells, the level of phosphorylated tif1β/ser was halved in megakaryocytic differentiated k consistent with the observation that tif1β promotes cell cycle progression by the up-regulation of its ser phosphorylation, the amount of tif1β/ser phosphorylation may be utilized as a proliferation marker in cancer cells. discussion this investigation demonstrates that the phosphorylation of tif1β/ser by kinase may act as a molecular switch in the temporal regulation of gene expression. tif1β/ser is located close to the hp box, the pxvxl motif, which is responsible for the interaction between tif1β and hp and its phosphorylation negatively affects this interaction. the dynamic phosphorylation of tif1β/ser suggests that this event may regulate the functions of tif1β in cell cycle progression. the central question then is: how may the phosphorylation of tif1β/ser regulate tif1β-mediated gene expression? we addressed this question by investigating the potential regulatory functions of tif1β on key genes for cell cycle pregression and the effects of over-expressing phosphorylation-deficient or phosphomimetic mutants of tif1β/ser on cell cycle progression. tif1β is proposed to function as a universal corepressor protein for the krab zinc finger protein superfamily of transcriptional repressors. the recruitment of hp proteins by the krab-tif1β complex to specific loci within the genome through the formation of heterochromatin-like complexes may silence gene activity. the phosphorylation of tif1β/s compromises its interaction with hp1β in a manner that is related to cell cycle-regulated gene expression the disruption of the interaction between hp1β and tif1β by the phosphorylation of tif1β/ser suggests that this phosphorylation/dephosphorylation of tif1β/ser may be the means of regulation of tif1β- and hp1β-mediated gene silencing. the results of chip experiments suggest that the majority of tif1β associated with the promoters of cyclin a in g phase cells is likely to be unphosphorylated tif1β/ser this conclusion is supported by two lines of evidence: very low levels of phosphorylated tif1β/ser were observed in g cells, and overexpressed flag-tif1β/s473a bound to the promoters of cdc and cdc25a better than flag-tif1β/s473e. when cells were released into the s phase, the association of unphosphorylated tif1β/ser with these promoters decreased, accompanying an increased level of phosphorylated tif1β/ser the dynamics of tif1β/ser phosphorylation and tif1β-binding to the cyclin a promoter indicate that un-phosphorylated tif1β/ser is responsible for silencing the cyclin a gene in the g phase. the observation that hp1β interacted strongly with un-phosphorylated tif1β/ser is consistent with the chip results concerning the over-expressed recombinant variants, where over-expressed flag-tif1β/s473a associated better with the promoter region of cdc or cdc25a than that did flag-tif1β/s473e. the treatment of cells with cyclin a sirna led to an accumulation of cells in prophase and mitosis to a degree similar to that observed for cyclin b consistent with the requirement of cyclin a for g1/s and g2/m transitions. interestingly, the chip results and the effects of over-expressed flag-tif1β/ser473a on cell cycle progression are consistent with published results for the sirna knockdown of cyclin a. hp1β recruitment to e2f-binding element of cdc and cdc25a promoters was affected by the phosphorylation state of tif1β/ser the level of hp1β recruitment to cdc or cdc25a promoter was increased when wild-type flag-tif1β or flag-tif1β/s473a were ectopically expressed. this association was compromised by the phosphomimetic mutant, s473e, which suggests that hp1β recruitment is negatively regulated by phosphorylation of tif1β/ser likewise, ectopically expressed hp1β resulted in elevated recruitment of wild-type flag-tif1β or flag-tif1β/s473a. these observations provide a clue that the recruitment of tif1β and hp1β could work in a positive feedback manner. the above results demonstrate that the dynamic interconversion between unphosphorylated and phosphorylated forms of tif1β/ser may be crucial in the regulation of gene expression during cell cycle progression. despite extensive efforts to perform chip with rabbit anti-phosphorylated tif1β/ser antibody, no significant result was obtained. a likely explanation of this failure is that phosphorylated tif1β/ser is not associated with the promoter of cyclin a in the g phase or, alternatively, the epitope in the multiprotein complex is masked from antibody access. the way in which tif1β disassociates from the hp1-containing heterochromatin is unclear. however, the findings herein provide a molecular explanation, which involves pkc-mediated phosphorylation at tif1β/ser. phosphorylated tif1β/s level may serve as a proliferation marker tif1β transcriptional repression activity depends on the interaction between tif1β and hp1β. this interaction is essential to the relocation of tif1β from euchromatin to heterochromatin that accompanies the differentiation of primitive endoderm-like cells. tif1β is known to interact differentially with hp1β and hp1γ in differentiated and non-differentiated cells. in non-differentiated cells, tif1β/hp interaction occurs only in euchromatin and selectively involves hp1β and hp1γ, but not hp1α. in differentiated cells, on the other hand, tif1β selectively associates with hp1β in heterochromatin, while tif1β and hp1γ interaction occurs only in euchromatin. these conclusions agree with the reduced level of phosphorylated tif1β/ser seen here in differentiated k cells. the results herein also revealed that un-phosphorylated tif1β/ser interacts more strongly with hp1β than its phosphorylated counterpart. these results further suggest that the phosphorylation of tif1β/ser regulates the differential interaction between tif1β and hp1β. what may be the functional consequences of the phosphorylation of tif1β/ser and its consequent dissociation from hp1s must be addressed. tif1β interacts with e2f, trip-br and cbp/p and potentiates the co-activation of e2f-1/dp- by trip-br protein. phosphorylated tif1β/ser may thus be involved in this tripartite functional interaction. this suggestion is supported by our findings that the induction of cyclin a is accompanied by an increased level of phosphorylated tif1β/ser and reduced binding of tif1β to the promoter. a most provocative question that remains to be answered is whether the ser473-phosphorylated tif1β may interact preferentially with transcription factors and serve as a coactivator. the results also demonstrated that the level of phosphorylated tif1β/ser peaks at early s- and m-phases. two phosphorylation sites other than ser ser and ser were also identified in the course of this investigation. both sites are located in the bromodomain of tif1β. the phosphorylation-deficient or phosphomimetic mutants of ser did not influence the phosphorylation of ser, suggesting that these phosphorylations may be independent events during mitosis. other phosphorylation sites important for regulating the function of tif1β have also been identified. the functional consequences of ser and ser phosphorylation remain to be investigated. pkc mediated tif1β/s phosphorylation may be involved in cell cycle progression numerous examples have demonstrated that pkcδ is centrally involved in cell proliferation and differentiation. the activation of pkcδ uniquely mediates insulin-induced proliferation: pkcδ is activated by insulin and interacts with insulin receptor and irs. insulin-activated pkcδ interacts with 3-phosphoinositide-dependent protein kinase to regulate protein kinase b and is responsible for stat activation and keratinocyte proliferation. although the over-expressed pkcδ is mainly located in the cytoplasm, it has been shown here and by kajimoto et al. to partially localize to the nucleus. the observation herein that the serum-stimulated phosphorylation of tif1β/ser correlates strongly with g1/s progression and cyclin a expression uncovers a novel mechanism of pkcδ-mediated g to s phase cell cycle progression. phosphorylation of tif1β/ser gene activation and stem cell proliferation the dynamic phosphorylation of tif1β/ser during cell proliferation and differentiation suggests that phosphorylation/dephosphorylation is crucial for regulating the transcriptional activity of tif1β. during the differentiation of embryonic carcinoma cells, the intracellular distribution of tif1β changes from diffuse nuclear staining to discrete foci and colocalizes with heterochromatin. the steady-state level of tif1β is also decreased. the reduced levels of phosphorylated tif1β/ser as well as the lower steady-state tif1β levels in differentiated cells suggest that tif1β may be mainly localized to heterochromatin in differentiated cells. in embryonic stem cells, tif1β is present in complexes with various pluripotent markers, including rex- dax- and nanog. since phosphorylated tif1β/ser seems to be preferentially associated with cell proliferation, it is important to determine whether it resides in these complexes. in fact, the level of phosphorylated tif1β/ser may serve as a proliferation marker. the epigenetic silencing of retrovirus transcription is caused by the binding of a tif1β corepressor complex to retrovirus primer binding site. the likely recruitment of tif1β by a dna-binding krab-box containing zinc finger protein for pbs-mediated silencing should be addressed, and the question of whether the regulation of the phosphorylation of tif1β/ser or the formation of tif1β corepressor complex is participating in retrovirus transcription should also be investigated.  although the tif1β co-repressor function is known to be related to hp1β, few studies have addressed the specific gene targets of tif1β. we have found that cell cycle progression is regulated by tif1β. the key cell cycle regulatory genes, cyclin a cdc and cdc25a are targeted by tif1β, and phosphorylation of tif1β/ser is associated with the activation of these genes. tif1β/s phosphorylation is up-regulated during the s phase in hela cells and the interaction between hp1β and tif1β is compromised when ser is phosphorylated. these observations suggest that the phosphorylation of tif1β/ser may regulate gene expression by abolishing its interaction with hp1β. thus, phosphorylation of tif1β/ser plays a crucial role in epigenetic regulation of gene expression. 
the interaction between tif1β and hp is crucial for heterochromatin formation and maintenance. finally, we found that the phosphorylation of tif1β/ser is mediated by the pkcδ pathway and is closely linked to cell proliferation. the modulation of hp1β-tif1β interaction through the phosphorylation/de-phosphorylation of tif1β/ser may constitute a molecular switch that regulates the expression of particular genes. this work demonstrates that tif1β is phosphorylated on ser the alteration of which is dynamically associated with cell cycle progression and functionally linked to transcriptional regulation. higher levels of phosphorylated tif1β/ser may be associated with the expression of key regulatory genes for cell cycle progression and the proliferation of cells. consistent with possible inhibition of tif1β's corepressor function, the phosphorylation of the ser residue, which is located near the hp1-interacting pxvxl motif, compromised the formation of tif1β-hp complex. as an epigenetic regulator, the transcriptional intermediary factor 1β /kap1/trim28) has been linked to gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein family and various other chromatin factors.
 as an epigenetic regulator, the transcriptional intermediary factor 1β /kap1/trim28) has been linked to gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein family and various other chromatin factors. the interaction between tif1β and hp is crucial for heterochromatin formation and maintenance. the hp1-box, pxvxl, of tif1β is responsible for its interaction with hp however, the underlying mechanism of how the interaction is regulated remains poorly understood.  this work demonstrates that tif1β is phosphorylated on ser the alteration of which is dynamically associated with cell cycle progression and functionally linked to transcriptional regulation. phosphorylation of tif1β/ser coincides with the induction of cell cycle gene cyclin a at the s-phase. interestingly, chromatin immunoprecipitation demonstrated that the promoter of cyclin a gene is occupied by tif1β and that such occupancy is inversely correlated with ser phosphorylation. additionally, when hp1β was co-expressed with tif1β/s473a, but not tif1β/s473e, the colocalization of tif1β/s473a and hp1β to the promoters of cdc and cdc25a was enhanced. non-phosphorylated tif1β/ser allowed greater tif1β association with the regulatory regions and the consequent repression of these genes. consistent with possible inhibition of tif1β's corepressor function, the phosphorylation of the ser residue, which is located near the hp1-interacting pxvxl motif, compromised the formation of tif1β-hp complex. finally, we found that the phosphorylation of tif1β/ser is mediated by the pkcδ pathway and is closely linked to cell proliferation.  the modulation of hp1β-tif1β interaction through the phosphorylation/de-phosphorylation of tif1β/ser may constitute a molecular switch that regulates the expression of particular genes. higher levels of phosphorylated tif1β/ser may be associated with the expression of key regulatory genes for cell cycle progression and the proliferation of cells. 