BACKGROUND
the primary catalytic function of ppiases  is to accelerate cis-trans isomerization of xaa-pro peptide bonds within polypeptide chains  <cit> .
hence, a role for peptidyl-prolyl cis/trans isomerases  in the folding of newly synthesized proteins was inferred and chaperone-like activity has been associated with several ppiases  <cit> .
there are few classes among the ppiase family such as the cyclophilins   <cit> , the fk506-binding proteins   <cit>  and the prototyping protein phosphatase 2a  phosphatase activator   <cit> .
par <dig> and its isoform par <dig> belong to a class of ppiases called parvulins.
both proteins are encoded by the gene pin <dig> which is located on chromosome xq <dig>  within the human genome  <cit> .
the pin <dig> promoter is tata-less and situated in a cpg island typical of housekeeping genes.
human par <dig> has a molecular weight of  <dig> kda  <cit>  and consists of  <dig> amino acids.
it was first cloned as a homologue of human pin <dig> and e. coli par <dig>  <cit> .
par <dig> has an n-terminal basic region  and a c-terminal ppiase domain   <cit> .
par <dig> is an elongated isoform of par <dig> that resulted by alternative transcription initiation  <cit> .
whereas par <dig> is already present in metazoans, par <dig> is only expressed in cells of great apes, but not in those of other primates  <cit> .
in mammalian cells, the expression of par <dig> was up-regulated in heart and skeletal muscles  <cit> .
functional studies have shown that both par <dig> and par <dig> are capable of accelerating the build-up of microtubules  <cit> .
studies in hepg <dig> cells and mouse liver revealed that par <dig> associates with insulin receptor substrate  <dig>  via its n-terminal residues, thereby enhancing insulin-induced irs- <dig> phosphorylation and affecting glucose metabolism  <cit> .
immunocytochemistry studies using a par <dig> antibody have shown that endogenous protein is accumulated in the nucleoli of cells associating with pre-ribosomal complexes  <cit> .
from cellular fractionation and mutational studies, par <dig> showed an uneven distribution pattern between the cytosol and the nucleus fraction  <cit> , and was 2-fold higher in the nucleus than in the cytoplasm.
based on homologies and similarities of par <dig> to members of the high mobility group  of proteins  <cit>  and with structural features within the catalytic domain similar to the transcription factor lef- <dig>  <cit> , double-stranded dna constructs were developed and tested for their par <dig> binding affinity in fluorescence titration, dna-cellulose binding and electro-mobility shift assays.
par <dig> bound preferentially in a 100– <dig> nm range to bent at-rich dna octamers in vitro.
such bent at-rich segments of dna are supposed to dictate nucleosome positioning  <cit>  and play a role in transcription initiation.
experiments with truncated par <dig> showed that the unstructured basic n-terminal part with sequence similarity to the chromatin-unfolding domain of hmgn proteins was indispensable for high affinity dna binding.
subsequent experiments demonstrated that the phosphorylation of par <dig> on ser <dig> regulated its sub-cellular localization to the nucleus and its ability to bind dna in vitro  <cit> .
mutation of ser <dig> to ala abolishes phosphorylation and alters the sub-cellular localization of par <dig> from predominantly nuclear to significantly cytoplasmic.
immunostaining showed that a glu <dig> mutant of par <dig>  which mimics the phosphorylated state of ser <dig>  does not penetrate into the nucleoplasm.
as opposed to wild-type par <dig>  the in vitro dna binding affinity of the glu <dig> mutant was strongly reduced, indicating that only the de-phosphorylated protein is active in dna-binding in the cellular nucleus.
a considerable body of evidence supported the fact that par <dig> is a nuclear protein and binds to double stranded dna in vitro.
in addition par <dig> was reported in immunofluorescence studies to accumulate around chromosomes during mitosis  <cit> .
moreover, pin <dig>  a paralogous protein to par <dig>  is a nuclear ppiase and it has been reported to be involved in the regulation of the cell cycle.
in the light of these evidences, it was of paramount importance to investigate the sub-nuclear localization of par <dig> by means of biochemical fractionation and to investigate its regulation within the cell cycle making use of cell cycle synchronization deprivation and qrt-pcr.
RESULTS
par <dig> is a non-histone chromatin protein
a small-scale biochemical fractionation procedure of an asynchronous culture of hela cells was performed .
subsequently, soluble cytosolic proteins , chromatin proteins with and without dnasei  and nuclear matrix proteins with and without dnasei  were isolated.
together with total cell extract  and recombinant par <dig>  these fractions were run on a sds-page gel, which was further analyzed by western blotting and densitometry .
the band area signals for par <dig> in the chromatin fractions s2+ and s2- were 3- and 2-fold higher, respectively, than those of the nuclear matrix fractions s3+ and s3-.
the densitogram shows the distribution of par <dig> in the various fractions .
from the par <dig> band signal in s2+  it is deduced that par <dig> was  <dig> -fold released from chromatin when treated with dnase i as opposed to s2-, which did not undergo any nuclease treatment.
hence, there is an enrichment of par <dig> in the chromatin fractions when compared to the nuclear matrix fractions.
therefore, the differential release of par <dig> from chromatin after dnase i treatment, lends evidence to its intimate association with dna.figure  <dig>
par <dig> localizes more to chromatin than to nuclear matrix.
a) an asynchronous culture of hela cells was subjected to biochemical fractionation.
b) immunoblotting with the affinity-purified αppiase, αmek <dig>  αlamin b <dig> and αhistone h <dig> antisera to benchmark the different fractions at a 1: <dig> dilution.
c) distribution of par <dig> in the various fractions quantified by densitometric analysis.
the area of the band signals from three independent experiments were measured, normalized and the background signals were subtracted.
multi gauge densitometry software version  <dig>  was used.
par <dig> binds to dna in vivo
in concert with our observation that par <dig> associates with chromatin and is released after treatment with dnase i, it was mandatory to verify the in vivo binding of par <dig> to dna using a different biochemical fractionation approach.
qproteome nuclear subfractionation kit from qiagen  was used whereby nuclear proteins are separated to allow for the detection of low-abundance proteins such as transcription factors.
fractions of cytosolic proteins , nucleic-acid binding proteins  and benzonase solubilized nuclei  were isolated.
eluates were collected using increasing sodium salt concentrations .
fraction s <dig> contained nucleic acid-binding proteins  that was later bound on a phospho-cellulose column.
fractions s <dig>  e <dig> - e <dig> eluted with  <dig> ,  <dig> and  <dig> m nacl), s4+ together with tce and rhpar <dig> were run on a sds-page gel.
analysis of the eluates on an immunoblot showed that at  <dig>  m  and  <dig> m  par <dig> was eluted .
this finding demonstrated that par <dig> was enriched in the fraction that was known as nucleic acid-binding.
thus, par <dig> was bound to dna when isolated from an endogenous cellular environment.
s4+ was the supernatant of the nuclei pellet p4+ that was treated with benzonase, and it was referred to as the solubilized nuclei fraction.
the presence of par <dig> in s <dig>  and s4+  went to validate previously published data that assigned par <dig> to these compartments  <cit> .figure  <dig>
par <dig> binds to dna
in vivo
and elutes with increasing salt concentrations.
a) an asynchronous culture of hela cells was subjected to a biochemical fractionation of nuclear and nucleic acid binding proteins.
b) immunoblotting with the affinity-purified αppiase, mek <dig>  and nuclear matrix protein p <dig> antisera to benchmark the different fractions at 1: <dig> dilution.
par <dig> and polytene chromosomes
we have shown with ample credibility that par <dig> is a chromatin and non-histone protein, which binds to dna in vivo.
our next approach to validate this finding was to investigate if par <dig> in drosophila melanogaster is associated with chromosomes.
human par <dig>  shares  <dig> % sequence identity with its ortholog in drosophila melanogaster  and within their ppiase domains, they share 81% sequence identity .
based on this fact, western blots were carried out using the nuclear extracts  of kc cells from drosophila melanogaster.
a protein band of  <dig> kda was detected when the αppiase was used and this band was absent when the pre-immune antiserum of αppiase was applied , proofing that the par <dig> ortholog is associated with polytene chromosomes.
as we have now shown that hpar <dig> binds to condensed and non-condensed dna in vitro and par <dig> is phosphorylated by the casein kinase  <dig>  a serine/threonine-directed kinase implicated in cell cycle control  <cit> , we wondered whether hpar <dig> might undergo cell cycle correlated expression levels.figure  <dig>
par <dig> is found on the nuclear extracts of
drosophila melanogaster
.
a) sequence alignment of human par <dig> with that of drosophila melanogaster.
identical residues are shown in blue.
multiple sequence alignment with the clustal series of programs was used b) immunoblots of nuclear extracts from drosophila melanogaster kc cells, recombinant par <dig>  and total cell extract .
lanes  <dig> and 2: nuclear extract from drosophila melanogaster kc cells; lanes  <dig> and 4: rhpar <dig> and tce.
a band of  <dig> kda is observed on lanes  <dig>   <dig> and  <dig> and not on lane  <dig> that corresponded to the molecular weight of par <dig>
par <dig> is up-regulated during the s and g2/m phases of the cell cycle
the transcriptional regulation of par <dig> across the cell cycle was studied with the aid of qrt-pcr technology.
human foreskin fibroblasts  were suitable for cell cycle synchronization by serum deprivation.
after 30– <dig> hrs of starvation by depriving cultured hffs from fcs, an aliquot of cells was obtained and termed g <dig> cells at t =  <dig>  the rest of the cells were re-stimulated to enter the cell cycle.
aliquots of cells were collected within  <dig>   <dig>  and  <dig> hrs after growth re-stimulation for g <dig>  s and g2/m cells respectively  <cit> .
cell cycle phases were determined by dna staining and fluorescence-activated cell sorting  analysis.
the distribution of the cells relative to their location during the cell cycle can be seen figure 4a.
quantitative rt-pcr analyses were done  using primers for par <dig> as previously described  <cit> .
u <dig> splisosomal rna  was used as an internal control  <cit>  and transcription of cyclin b <dig> served as a marker gene for the g2/m phase  <cit> .
transcriptional expression profiles for par <dig> and cyclin b <dig> were normalized against the expression profile for snrna u <dig>  these results showed that par <dig> is slightly but significantly up-regulated 2- and 3-fold in the s and g2/m phases of the cell cycle, respectively, where dna duplication and cell division takes place.
to examine, whether the observed transcriptional cell cycle dependence of hpar <dig> is reflected on the protein level, we additionally performed western blot studies followed by subsequent densitometric analysis.
par <dig> is translationally up-regulated within the g <dig>  s and g2/m phases of the cell cycle  with respect to the g <dig> phase.
the protein shows a ~3-fold increase in g <dig> and ~4-fold increase in both s and g2/m phases, respectively .figure  <dig>
up-regulation of par <dig> in the s and g2/m phases.
a) percentage cell cycle distribution.
human foreskin fibroblasts  were examined by dna staining with propidium iodide followed by facs analysis.
numbers denote percentage of cells in g <dig>  g <dig>  s and g2/m phase.
percentage of cells at g <dig> was fixed to  <dig> after serum deprivation.
the arithmetic mean , is an indicator of the fluorescence intensity i.e.
the number of propidium iodide molecules bound to dna.
a value above  <dig> is considered as appropriate.
the results show the average of five independent cell cycle synchronisation experiments.
for each sample,  <dig> ×  <dig> cells were analysed b) qrt-pcr analysis of par <dig> across the cell cycle with synchronised hff.
white columns represent the percentage mrna expression, dark blue columns the percentage mrna expression of cyclin b <dig>  after 30– <dig> hrs of serum deprivation, cells in g <dig> were arrested at t =  <dig> h. hff cells were re-stimulated with 20% fcs.
g <dig> cells were harvested after t =  <dig> h, s phase cells after t =  <dig> h and g2/m cells after t =  <dig> hrs.
mrna levels of par <dig> were normalised using snrna u <dig>  one-way anova was performed using r. the bars represent the standard deviation from five independent cell cycle synchronisation and qrt-pcr experiments conducted.
the asterisks on the bars indicate the statistical significance expressed in p-values .
c) translational up-regulation of protein across the cell cycle.
western blot analysis was done using αppiase  and β- actin  antisera.
<dig> μg of protein for each cell lysate from the respective phase of the cell cycle was loaded.
d) densitometric analysis from three independent cell cycle synchronisation and western blot experiments of par <dig> protein.
band signals were normalised with β- actin.
discussion
it has been shown that par <dig> is enriched in the nucleus and that it has sequence and structural identities to hmgb and hmgn proteins.
the dna and chromatin association demonstrated in this study appear to be even more plausible when combining these findings with the functions of a series of recently discovered dna-associated parvulin binding partners.
ascc <dig>  a member of a novel transcription co-activator complex  <cit> , was reported to interact with pin <dig> in a yeast two-hybrid screening  <cit> .
in the same assay, polycomb group  protein ezh <dig> , a histone methyltransferase associated with transcriptional repression, was reported as an interactor to pin <dig>  fujiyama and colleagues demonstrated that nucleolin interacted with par <dig>  <cit> .
in co-immunoprecipitation experiments, rickards and co-authors confirmed nucleolin to be associated with chromatin-containing rrna genes transcribed by rna polymerase i only  <cit> .
they proposed that the function of nucleolin is to permit the transcription of nucleolar chromatin by rna polymerase i. furthermore, nucleolin was reported as a nuclear protein with a histone chaperone function  <cit> .
its histone chaperone activity enhanced the activity of chromatin remodeling machineries such as swi/snf and acf.
with these several lines of evidence shown, we postulate that par <dig> is involved in transcriptional regulation and/or chromatin remodeling based on its association to chromatin.
beside par <dig> other proline isomerases have also been shown to be associated with chromatin.
the nuclear fkbp, spfkbp39p from schizosaccharomyces pombe was reported to be a histone chaperone regulating rdna silencing and it influenced chromatin organization both in vivo and in vitro  <cit> .
still in yeast, fpr <dig>  a member of the fkbps in saccharomyces cerevisiae was shown to bind the amino-terminal tail of histones h <dig> and h <dig> and catalyzes the isomerization of histone h <dig> proline p <dig> and p <dig> in vitro  <cit> .
in the same work, they also showed that the abrogation of fpr <dig> catalytic activity in vivo resulted in increasing levels of h3k <dig> methylation and delayed transcriptional induction kinetics of yeast-specific genes.
the work summarizes proline isomerization as a novel non-covalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.
above all, pin <dig> and its ortholog ess <dig>  have been demonstrated to modulate the c-terminal domain  of rna polymerase ii  during the transcriptional cycle  <cit> .
the ctd is the largest subunit of pol ii and contains repeatedly the heptad sequence  that plays a key role in the transcription cycle, coordinating the exchange of transcription and rna processing factors.
ctd exhibits several structurally flexible heptapeptide repeats that undergo conformational changes in the advent of serine phosphorylation and proline isomerization by pin1/ess <dig>  which then have an effect on the transcription of genes.
the up-regulation of par <dig> in the s and g2/m phases of the cell cycle in concert with its dna and chromatin association might be a hint that par <dig> also acts on the transcriptional regulation of genes.
CONCLUSIONS
to obtain valuable clues for its cellular function it was of paramount importance to investigate the sub-nuclear localization of par <dig>  the performed analysis of the nuclear fraction separating chromatin and nuclear matrix proteins ascertains that par <dig> is a non-histone protein associated with chromatin.
previous studies from dna-cellulose binding assays and emsa have demonstrated that par <dig> binds to dna in vitro.
our results from the fractionation of nuclear and nucleic acid-binding proteins strengthen the contention that par <dig> binds to dna in vivo.
in addition par <dig> was reported in immunofluorescence studies to accumulate around chromosomes during mitosis.
we have now demonstrated that par14’s ortholog in drosophila melanogaster is associated with the polytene chromosomes and that hpar <dig> can be collected from a chromatin protein fraction in human cells.
the transcriptional and translational up-regulation of par <dig> in the s and g2/m phases of the cell cycle and the dependence of its dna binding properties on the phosphorylation by the casein kinase  <dig> relates the action of par <dig> to cell cycle regulation and/or dna repair.
in concert with its dna and chromatin association and the homologies of structural motifs of par <dig> to those of the transcription factor lef- <dig> these findings suggest that par <dig> parallels in part the cellular function of its paralog pin <dig>  but is acting on non-phosphorylated epitopes of its target proteins.

 the primary catalytic function of ppiases is to accelerate cis-trans isomerization of xaa-pro peptide bonds within polypeptide chains. hence, a role for peptidyl-prolyl cis/trans isomerases in the folding of newly synthesized proteins was inferred and chaperone-like activity has been associated with several ppiases. there are few classes among the ppiase family such as the cyclophilins, the fk506-binding proteins and the prototyping protein phosphatase 2a phosphatase activator. par and its isoform par belong to a class of ppiases called parvulins. both proteins are encoded by the gene pin which is located on chromosome xq within the human genome. the pin promoter is tata-less and situated in a cpg island typical of housekeeping genes. human par has a molecular weight of kda and consists of amino acids. it was first cloned as a homologue of human pin and e. coli par. par has an n-terminal basic region and a c-terminal ppiase domain. par is an elongated isoform of par that resulted by alternative transcription initiation. whereas par is already present in metazoans, par is only expressed in cells of great apes, but not in those of other primates. in mammalian cells, the expression of par was up-regulated in heart and skeletal muscles. functional studies have shown that both par and par are capable of accelerating the build-up of microtubules. studies in hepg cells and mouse liver revealed that par associates with insulin receptor substrate via its n-terminal residues, thereby enhancing insulin-induced irs- phosphorylation and affecting glucose metabolism. immunocytochemistry studies using a par antibody have shown that endogenous protein is accumulated in the nucleoli of cells associating with pre-ribosomal complexes. from cellular fractionation and mutational studies, par showed an uneven distribution pattern between the cytosol and the nucleus fraction, and was 2-fold higher in the nucleus than in the cytoplasm. based on homologies and similarities of par to members of the high mobility group of proteins and with structural features within the catalytic domain similar to the transcription factor lef-, double-stranded dna constructs were developed and tested for their par binding affinity in fluorescence titration, dna-cellulose binding and electro-mobility shift assays. par bound preferentially in a 100– nm range to bent at-rich dna octamers in vitro. such bent at-rich segments of dna are supposed to dictate nucleosome positioning and play a role in transcription initiation. experiments with truncated par showed that the unstructured basic n-terminal part with sequence similarity to the chromatin-unfolding domain of hmgn proteins was indispensable for high affinity dna binding. subsequent experiments demonstrated that the phosphorylation of par on ser regulated its sub-cellular localization to the nucleus and its ability to bind dna in vitro. mutation of ser to ala abolishes phosphorylation and alters the sub-cellular localization of par from predominantly nuclear to significantly cytoplasmic. immunostaining showed that a glu mutant of par which mimics the phosphorylated state of ser does not penetrate into the nucleoplasm. as opposed to wild-type par the in vitro dna binding affinity of the glu mutant was strongly reduced, indicating that only the de-phosphorylated protein is active in dna-binding in the cellular nucleus. a considerable body of evidence supported the fact that par is a nuclear protein and binds to double stranded dna in vitro. in addition par was reported in immunofluorescence studies to accumulate around chromosomes during mitosis. moreover, pin a paralogous protein to par is a nuclear ppiase and it has been reported to be involved in the regulation of the cell cycle. in the light of these evidences, it was of paramount importance to investigate the sub-nuclear localization of par by means of biochemical fractionation and to investigate its regulation within the cell cycle making use of cell cycle synchronization deprivation and qrt-pcr.  par is a non-histone chromatin protein a small-scale biochemical fractionation procedure of an asynchronous culture of hela cells was performed. subsequently, soluble cytosolic proteins, chromatin proteins with and without dnasei and nuclear matrix proteins with and without dnasei were isolated. together with total cell extract and recombinant par these fractions were run on a sds-page gel, which was further analyzed by western blotting and densitometry. the band area signals for par in the chromatin fractions s2+ and s2- were 3- and 2-fold higher, respectively, than those of the nuclear matrix fractions s3+ and s3-. the densitogram shows the distribution of par in the various fractions. from the par band signal in s2+ it is deduced that par was -fold released from chromatin when treated with dnase i as opposed to s2-, which did not undergo any nuclease treatment. hence, there is an enrichment of par in the chromatin fractions when compared to the nuclear matrix fractions. therefore, the differential release of par from chromatin after dnase i treatment, lends evidence to its intimate association with dna.figure par localizes more to chromatin than to nuclear matrix. a) an asynchronous culture of hela cells was subjected to biochemical fractionation. b) immunoblotting with the affinity-purified αppiase, αmek αlamin b and αhistone h antisera to benchmark the different fractions at a 1: dilution. c) distribution of par in the various fractions quantified by densitometric analysis. the area of the band signals from three independent experiments were measured, normalized and the background signals were subtracted. multi gauge densitometry software version was used. par binds to dna in vivo in concert with our observation that par associates with chromatin and is released after treatment with dnase i, it was mandatory to verify the in vivo binding of par to dna using a different biochemical fractionation approach. qproteome nuclear subfractionation kit from qiagen was used whereby nuclear proteins are separated to allow for the detection of low-abundance proteins such as transcription factors. fractions of cytosolic proteins, nucleic-acid binding proteins and benzonase solubilized nuclei were isolated. eluates were collected using increasing sodium salt concentrations. fraction s contained nucleic acid-binding proteins that was later bound on a phospho-cellulose column. fractions s e - e eluted with, and m nacl), s4+ together with tce and rhpar were run on a sds-page gel. analysis of the eluates on an immunoblot showed that at m and m par was eluted. this finding demonstrated that par was enriched in the fraction that was known as nucleic acid-binding. thus, par was bound to dna when isolated from an endogenous cellular environment. s4+ was the supernatant of the nuclei pellet p4+ that was treated with benzonase, and it was referred to as the solubilized nuclei fraction. the presence of par in s and s4+ went to validate previously published data that assigned par to these compartments.figure par binds to dna in vivo and elutes with increasing salt concentrations. a) an asynchronous culture of hela cells was subjected to a biochemical fractionation of nuclear and nucleic acid binding proteins. b) immunoblotting with the affinity-purified αppiase, mek and nuclear matrix protein p antisera to benchmark the different fractions at 1: dilution. par and polytene chromosomes we have shown with ample credibility that par is a chromatin and non-histone protein, which binds to dna in vivo. our next approach to validate this finding was to investigate if par in drosophila melanogaster is associated with chromosomes. human par shares % sequence identity with its ortholog in drosophila melanogaster and within their ppiase domains, they share 81% sequence identity. based on this fact, western blots were carried out using the nuclear extracts of kc cells from drosophila melanogaster. a protein band of kda was detected when the αppiase was used and this band was absent when the pre-immune antiserum of αppiase was applied, proofing that the par ortholog is associated with polytene chromosomes. as we have now shown that hpar binds to condensed and non-condensed dna in vitro and par is phosphorylated by the casein kinase a serine/threonine-directed kinase implicated in cell cycle control, we wondered whether hpar might undergo cell cycle correlated expression levels.figure par is found on the nuclear extracts of drosophila melanogaster. a) sequence alignment of human par with that of drosophila melanogaster. identical residues are shown in blue. multiple sequence alignment with the clustal series of programs was used b) immunoblots of nuclear extracts from drosophila melanogaster kc cells, recombinant par and total cell extract. lanes and 2: nuclear extract from drosophila melanogaster kc cells; lanes and 4: rhpar and tce. a band of kda is observed on lanes and and not on lane that corresponded to the molecular weight of par par is up-regulated during the s and g2/m phases of the cell cycle the transcriptional regulation of par across the cell cycle was studied with the aid of qrt-pcr technology. human foreskin fibroblasts were suitable for cell cycle synchronization by serum deprivation. after 30– hrs of starvation by depriving cultured hffs from fcs, an aliquot of cells was obtained and termed g cells at t =  the rest of the cells were re-stimulated to enter the cell cycle. aliquots of cells were collected within and hrs after growth re-stimulation for g s and g2/m cells respectively. cell cycle phases were determined by dna staining and fluorescence-activated cell sorting analysis. the distribution of the cells relative to their location during the cell cycle can be seen figure 4a. quantitative rt-pcr analyses were done using primers for par as previously described. u splisosomal rna was used as an internal control and transcription of cyclin b served as a marker gene for the g2/m phase. transcriptional expression profiles for par and cyclin b were normalized against the expression profile for snrna u these results showed that par is slightly but significantly up-regulated 2- and 3-fold in the s and g2/m phases of the cell cycle, respectively, where dna duplication and cell division takes place. to examine, whether the observed transcriptional cell cycle dependence of hpar is reflected on the protein level, we additionally performed western blot studies followed by subsequent densitometric analysis. par is translationally up-regulated within the g s and g2/m phases of the cell cycle with respect to the g phase. the protein shows a ~3-fold increase in g and ~4-fold increase in both s and g2/m phases, respectively.figure up-regulation of par in the s and g2/m phases. a) percentage cell cycle distribution. human foreskin fibroblasts were examined by dna staining with propidium iodide followed by facs analysis. numbers denote percentage of cells in g g s and g2/m phase. percentage of cells at g was fixed to after serum deprivation. the arithmetic mean, is an indicator of the fluorescence intensity i.e. the number of propidium iodide molecules bound to dna. a value above is considered as appropriate. the results show the average of five independent cell cycle synchronisation experiments. for each sample, × cells were analysed b) qrt-pcr analysis of par across the cell cycle with synchronised hff. white columns represent the percentage mrna expression, dark blue columns the percentage mrna expression of cyclin b after 30– hrs of serum deprivation, cells in g were arrested at t =  h. hff cells were re-stimulated with 20% fcs. g cells were harvested after t =  h, s phase cells after t =  h and g2/m cells after t =  hrs. mrna levels of par were normalised using snrna u one-way anova was performed using r. the bars represent the standard deviation from five independent cell cycle synchronisation and qrt-pcr experiments conducted. the asterisks on the bars indicate the statistical significance expressed in p-values. c) translational up-regulation of protein across the cell cycle. western blot analysis was done using αppiase and β- actin antisera. μg of protein for each cell lysate from the respective phase of the cell cycle was loaded. d) densitometric analysis from three independent cell cycle synchronisation and western blot experiments of par protein. band signals were normalised with β- actin. discussion it has been shown that par is enriched in the nucleus and that it has sequence and structural identities to hmgb and hmgn proteins. the dna and chromatin association demonstrated in this study appear to be even more plausible when combining these findings with the functions of a series of recently discovered dna-associated parvulin binding partners. ascc a member of a novel transcription co-activator complex, was reported to interact with pin in a yeast two-hybrid screening. in the same assay, polycomb group protein ezh, a histone methyltransferase associated with transcriptional repression, was reported as an interactor to pin fujiyama and colleagues demonstrated that nucleolin interacted with par. in co-immunoprecipitation experiments, rickards and co-authors confirmed nucleolin to be associated with chromatin-containing rrna genes transcribed by rna polymerase i only. they proposed that the function of nucleolin is to permit the transcription of nucleolar chromatin by rna polymerase i. furthermore, nucleolin was reported as a nuclear protein with a histone chaperone function. its histone chaperone activity enhanced the activity of chromatin remodeling machineries such as swi/snf and acf. with these several lines of evidence shown, we postulate that par is involved in transcriptional regulation and/or chromatin remodeling based on its association to chromatin. beside par other proline isomerases have also been shown to be associated with chromatin. the nuclear fkbp, spfkbp39p from schizosaccharomyces pombe was reported to be a histone chaperone regulating rdna silencing and it influenced chromatin organization both in vivo and in vitro. still in yeast, fpr a member of the fkbps in saccharomyces cerevisiae was shown to bind the amino-terminal tail of histones h and h and catalyzes the isomerization of histone h proline p and p in vitro. in the same work, they also showed that the abrogation of fpr catalytic activity in vivo resulted in increasing levels of h3k methylation and delayed transcriptional induction kinetics of yeast-specific genes. the work summarizes proline isomerization as a novel non-covalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization. above all, pin and its ortholog ess have been demonstrated to modulate the c-terminal domain of rna polymerase ii during the transcriptional cycle. the ctd is the largest subunit of pol ii and contains repeatedly the heptad sequence that plays a key role in the transcription cycle, coordinating the exchange of transcription and rna processing factors. ctd exhibits several structurally flexible heptapeptide repeats that undergo conformational changes in the advent of serine phosphorylation and proline isomerization by pin1/ess which then have an effect on the transcription of genes. the up-regulation of par in the s and g2/m phases of the cell cycle in concert with its dna and chromatin association might be a hint that par also acts on the transcriptional regulation of genes.  to obtain valuable clues for its cellular function it was of paramount importance to investigate the sub-nuclear localization of par the performed analysis of the nuclear fraction separating chromatin and nuclear matrix proteins ascertains that par is a non-histone protein associated with chromatin. previous studies from dna-cellulose binding assays and emsa have demonstrated that par binds to dna in vitro. our results from the fractionation of nuclear and nucleic acid-binding proteins strengthen the contention that par binds to dna in vivo. in addition par was reported in immunofluorescence studies to accumulate around chromosomes during mitosis. we have now demonstrated that par14’s ortholog in drosophila melanogaster is associated with the polytene chromosomes and that hpar can be collected from a chromatin protein fraction in human cells. the transcriptional and translational up-regulation of par in the s and g2/m phases of the cell cycle and the dependence of its dna binding properties on the phosphorylation by the casein kinase relates the action of par to cell cycle regulation and/or dna repair. in concert with its dna and chromatin association and the homologies of structural motifs of par to those of the transcription factor lef- these findings suggest that par parallels in part the cellular function of its paralog pin but is acting on non-phosphorylated epitopes of its target proteins. 
par is released from the chromatin fraction after treatment with dnase i and elutes at high nacl concentrations from the nucleic acid-binding fraction. by means of sub-nuclear biochemical fractionations, we demonstrate that cellular par is associated with chromatin 3-fold higher than with the nuclear matrix in vivo. in the light of our results, par can be described as an endogenous non-histone chromatin protein, which binds dna in vivo. using qrt-pcr and western blotting we demonstrate that par is up-regulated during the s and g2/m phases in synchronised human foreskin fibroblasts cells. par a member of the parvulin family of peptidyl-prolyl cis-trans isomerases that is involved in rrna processing, microtubule formation and the glucose metabolism and has been suggested to play a role in chromatin remodeling on basis of sequence and structural identities to hmg proteins.
 par a member of the parvulin family of peptidyl-prolyl cis-trans isomerases that is involved in rrna processing, microtubule formation and the glucose metabolism and has been suggested to play a role in chromatin remodeling on basis of sequence and structural identities to hmg proteins. par is enriched in the nucleus and binds to double-stranded dna in vitro.  by means of sub-nuclear biochemical fractionations, we demonstrate that cellular par is associated with chromatin 3-fold higher than with the nuclear matrix in vivo. par is released from the chromatin fraction after treatment with dnase i and elutes at high nacl concentrations from the nucleic acid-binding fraction. using qrt-pcr and western blotting we demonstrate that par is up-regulated during the s and g2/m phases in synchronised human foreskin fibroblasts cells.  in the light of our results, par can be described as an endogenous non-histone chromatin protein, which binds dna in vivo. we propose that par is involved in a dna-dependent activity such as transcription. keywords par14non-histone proteinchromatindna-bindingtranscriptionissue-copyright-statement© the author 2015 