l929 mouse fibroblasts and a1 mouse embryonic fibroblasts ( parp-1 ) were maintained as subconfluent culture in high glucose ( 4.5 g / liter ) dulbecco 's modified eagle 's medium , with 10% fetal calf serum ( cambrex corp . ) , 2 mm l - glutamine ( cambrex corp . ) , and 50 units / ml pen - strep ( cambrex corp . ) .
exponentially growing cultures were transfected by lipofectamine plus reagent ( invitrogen ) adopting the manufacturer 's protocol with the following plasmid expression vectors : pci - ctcf - his tag ( kindly provided by elena m. klenova , department of biological sciences , university of essex , uk ) for the overexpression of human ctcf , pci - empty vector as control , and pbabe - puro for selection of transfectants . to obtain hypomethylated dna ,
cells were cultivated for 72 h in standard medium containing 5 m 5-aza ( sigma ) .
total cell lysates were obtained by direct lysis of cells in ripa buffer ( 50 mm tris - hcl ( ph 8) , 150 mm nacl , 0.5% sodium deoxycholate , 0.1% sds , 1% nonidet p-40 , 1 mm edta ) .
nuclei were collected from trypsinized and phosphate - buffered saline - washed cells by centrifugation following incubation ( 30 min ) in isolation buffer containing 10 mm tris - hcl ( ph 7.9 ) , 4 mm mgcl2 , 1 mm edta , 0.5 mm di - thiothreitol , 0.25 mm sucrose , 1% triton x-100 .
nuclear fraction was lysed in ripa buffer , and protein concentration was determined using the bradford protein assay reagent ( bio - rad ) with bovine serum albumin ( promega ) as standard .
both buffers were supplemented with protease inhibitors ( complete edta - free , roche applied science ) .
equal protein amounts were subjected to 6% sds - page and blotted onto hybond - ecl nitrocellulose membranes ( amersham biosciences ) .
the antibodies employed were as follows : mouse monoclonal ab anti - parp-1 ( c210 , alexis biochemicals ) , mouse monoclonal ab anti - parp-2 ( 4g8 , alexis ) , mouse monoclonal ab anti - par ( 10 ha , trevigen ) , mouse monoclonal ab anti - dnmt1 ( imgenex ) , rabbit polyclonal ab anti - ctcf ( upstate ) , rabbit polyclonal ab anti - sp1 ( h-225 , santa cruz biotechnology ) , rabbit polyclonal ab anti - lamin b1 ( abcam ) , and goat anti - mouse and anti - rabbit horseradish peroxidase - conjugated antibodies ( santa cruz biotechnology ) .
ctcf expression in the baculovirus system  pvlh - ctcf vector , kindly provided by dr .
klenova ( department of biological sciences , university of essex ) , was co - transfected with the bd baculogold baculovirus dna ( bd biosciences ) into sf9 cells .
after the co - transfection , the recombinant baculoviruses were selected and amplified following the manufacturer 's instructions .
the baculovirus recombinant hctcf his - tagged protein was purified from infected sf9 cells using the pro - bond purification system ( invitrogen ) .
bvctcf was quantified by silver staining ( silverquest , invitrogen ) using bovine serum albumin as standard . in vitro parp-1 activity assay 
bvctcf ( 0.2 , 0.4 , or 0.8 pmol ) was incubated for 2 h at 30 c with 4 pmol of human recombinant parp-1 ( alexis ) in 200 l of poly(adp - ribosyl)ation buffer containing 50 mm tris - hcl ( ph 8.0 ) , 10 mm mgcl2 , 10 mm c2h6os , and 200 m nad , in the presence or absence of 200 ng of dnase i - activated dna or 20 m pj-34 ( sigma ) .
parp-1 automodification reactions with or without pj-34 ( 20 m ) were used as control .
reactions were terminated by 20% trichloroacetic acid precipitation , and adp - ribose synthesis was assayed by 6% sds - page followed by western blotting with anti adp - ribose antibody .
an analogous radiometric assay was performed incubating bvctcf ( 0.03 , 0.05 , 0.1 , 0.2 , 0.4 pmol ) for 2 h at 30 c with 1 pmol of human recombinant parp-1 in 50 l of poly(adp - ribosyl)ation buffer and in the presence of [ h]nad ( 0.3 ci ) . following 20% trichloroacetic acid precipitation ,
co - immunoprecipitation ( co - ip)nuclei obtained from l929 cells were lysed in ip buffer ( 50 mm tris - hcl ( ph 7.5 ) , 5 mm edta , 300 mm nacl , 1% nonidet p-40 , 1% triton x-100 ) supplemented with protease - inhibitors ( complete edta - free , roche applied science ) .
lysates ( 1.5 mg ) were pre - cleared with protein a-(for ip anti ctcf and anti parp-1 ) or g ( for ip anti dnmt1)-agarose beads ( upstate ) on a rotative shaker at 4 c for 2 h and 30 min .
pre - cleared lysates were incubated with specific antibodies ( rabbit polyclonal ab anti - ctcf , upstate ; mouse monoclonal ab anti - dnmt1 , imgenex ; and rabbit polyclonal ab anti - parp-1 , alexis ) and with normal rabbit or mouse igg ( santa cruz biotechnology ) on a rotative shaker at 4 c .
the agarose beads , previously saturated with bovine serum albumin ( 1 g/l ) overnight , were added to the lysate / ab solutions and incubated for 2 h on a rotative shaker at 4 c .
subsequently , beads were washed in ip buffer and boiled in sds - page sample buffer , and the eluted proteins were analyzed by sds - page and western blotting .
pulldown assay50 l of probond resin ( invitrogen ) was added to 1.2 ml of 1  native purification buffer ( probond ) in the presence or absence of 0.8 pmol of bvctcf recombinant his - tagged protein .
then the resin was collected and incubated with 4 pmol of human recombinant parp-1 ( alexis ) in 300 l of 1  native purification buffer for 3 h on a rotative shaker at 4 c .
after washing the resin with 1 native purification buffer , proteins were eluted from the beads by boiling them in sds - page sample buffer .
protein - protein interaction was analyzed by 6% sds - page and western blot analysis .
dna methyltransferase activity  equal amounts of nuclear lysates from cells overexpressing ctcf and relative controls ( 35 g ) were analyzed for the dna methyltransferase activity by the epiquik dna methyltransferase assay kit ( epigentek ) following the manufacturer 's conditions .
this assay was also carried out to check the effect of bvctcf ( 0.02 , 0.04 , and 0.08 pmol ) or of same amounts of in vitro poly(adp - ribosyl)ated parp-1 ( 25 ) on 0.08 pmol of human recombinant dnmt1 ( new england biolabs ) .
human recombinant parp-1 ( alexis ) ( 0.08 pmol ) was used as control . rna extraction and
total rna ( 1.8 g ) , purified by rneasy mini kit ( qiagen ) , was subjected to retrotranscription using superscript first - strand synthesis system ( invitrogen ) .
real time pcr  expression of mrna for parp-1 , parp-2 , and dnmt1 genes was measured by real time pcr using taq - man gene expression assays ( applied biosystems ) following the manufacturer 's protocol for the absolute standard curve method on icycler iq detection system ( bio - rad ) .
the standard curve was generated using 1:1 serial dilutions ( from 100 to 12.5 ng ) of cdna obtained from control at 24 h as reference .
the amount of parp-1 , parp-2 , and dnmt1 mrnas was calculated adopting the standard curve method , and normalization was carried out using hypoxanthine - guanine phosphoribosyltransferase ( hprt1 ) as internal control gene .
taqman gene expression assay ids for each set of primers and probe were as follows : mm00599763m1 ( dnmt1 ) ; mm00500154m1 ( parp-1 ) ; mm00456462m1 ( parp-2 ) ; and mm00446968m1 ( hprt1 ) . dna extraction and methyl - accepting ability assay 
dna was purified from cells using the dneasy tissue kit ( qiagen ) , according to the manufacturer 's instructions .
methyl - accepting ability assay was carried out in a final volume of 50 l of 10 mm tris - hcl ( ph 7.9 ) , 10 mm mgcl2 , 50 mm nacl , 1 mm dithiothreitol in the presence of 1 g of purified dna and 1 unit of bacterial sssi methylase ( new england biolabs ) , using as methyl donor 16 m s - adenosylmethionine plus 10 ci / ml of [ methyl - h]s - adenosylmethionine ( ge healthcare ; specific activity 7080 ci / mmol ) .
the reaction was incubated for 1 h at 37 c and was stopped at 60 c for 30 min after addition of 1% sds and 250 g / ml of proteinase k. the incorporation of labeled methyl groups was evaluated on purified dna in a beckman ls-6800 liquid scintillation spectrometer .
methylation - sensitive southern blot analysis  the dna methylation level of minor satellite and b1 repeats was evaluated by southern blot analysis .
dnas ( 2 g ) were digested with 40 units of mspi or hpaii restriction enzymes for 16 h at 37 c .
after 1.5% agarose gel electrophoresis , the digested dna was blotted on hybond - n nylon membrane ( amersham biosciences ) , and the presence of new hpaii cutting sites was evidenced by hybridization to 3-digoxigenin - labeled single strand synthetic oligonucleotides as probes .
labeling of probes and detection was performed using digoxigenin oligonucleotide 3-end labeling kit and digoxigenin luminescent detection kit ( roche applied science ) .
sequence of probes were as follows : for minor satellite repeats , 5-ggaaacatgataaaaaccacagtgtagaacatattagatgagtgagttacactgaaaaacacattcgttggaaacgggatttgtagaacagtgtatatcaatgagttacaatgagaaacatc-3 ( 26 ) ; for b1 5-agtgagttccaggacagccag-3 ( 27 ) .
oligos were made by custom primers synthesis service ( invitrogen ) . as positive control for dna demethylation , digestion was performed in parallel on dna from cells treated for 72 h with 5 m 5-aza .
in the first experimental stage , our attention was focused on describing what happens in l929 fibroblast cells following transient or semi - stable ectopic ctcf overexpression ( 24 ) ( supplemental fig .
s1a ) . to quantify the proliferative capacity of ctcf - overexpressing cells , population doublings over time were calculated .
we found that control cultures doubled in 1 day versus 3 days for the ctcf - overexpressing one .
cell growth diminished by about 50% at 72 h post - transfection ( supplemental fig .
s1b ) ; no substantial change in the distribution of the cell population among the different phases of the cell cycle was observed at this time despite the inhibition of cell growth ( supplemental fig .
clonogenic ability was also affected when analyzed at 48 h post - transfection of pci - ctcf plus pbabe - puro ( 1:20 ) and puromycin selection for 2 weeks . at this time
s1d ) . unlike previously reported data , ctcf ectopic overexpression increased the percentage of dead ( supplemental fig .
s1f ) .   figure 1.in vivo and in vitro experiments addressing the correlation between ctcf and poly(adp - ribosyl)ation .
a , nuclear lysates from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci were analyzed by sds - page and western blot using antibodies against adp - ribose polymers ( par ) and against the indicated proteins .
b , real time rt - pcr for parp-1 and parp-2 performed on rna samples from cells at 24 , 48 , and 72 h post - transfection of both pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
data are reported as mean  s.e . of the ratio of parp-1 or parp-2 mrna level to hprt1 mrna level within each sample calculated from a minimum of three experiments carried out in duplicate .
d , radiometric parp-1 assay carried out in the presence (  ) or in the absence (  ) of  activated dna  and in the presence of pj-34 (  ) . to exclude poly(adp - ribosyl)ation activity of recombinant ctcf
, 0.4 pmol of bvctcf were assayed in poly(adp - ribosyl)ation buffer (  ) .
f , pulldown assay with bvctcf ( his - tagged ) and recombinant parp-1 .   in vivo and in vitro experiments addressing the correlation between ctcf and poly(adp - ribosyl)ation .
a , nuclear lysates from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci were analyzed by sds - page and western blot using antibodies against adp - ribose polymers ( par ) and against the indicated proteins .
b , real time rt - pcr for parp-1 and parp-2 performed on rna samples from cells at 24 , 48 , and 72 h post - transfection of both pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
data are reported as mean  s.e . of the ratio of parp-1 or parp-2 mrna level to hprt1 mrna level within each sample calculated from a minimum of three experiments carried out in duplicate .
d , radiometric parp-1 assay carried out in the presence (  ) or in the absence (  ) of  activated dna  and in the presence of pj-34 (  ) . to exclude poly(adp - ribosyl)ation activity of recombinant ctcf
, 0.4 pmol of bvctcf were assayed in poly(adp - ribosyl)ation buffer (  ) .
the extent of poly(adp - ribosyl)ation was analyzed by western blots of nuclear proteins obtained from adherent cells ectopically overexpressing ctcf and the respective control cells ( fig .
the use of anti - par antibodies showed that ctcf overexpression induced parp activity , leading to poly(adp - ribosyl)ation of chromatin proteins , including parp-1 .
anti - ctcf antibodies allowed the identification of the poly(adp - ribosyl)ated form of ctcf as a band running at 180 kda ( fig . 1a , top panel )
; it was impossible to detect this band using the anti - adp - ribose polymer antibodies because the smearing of polymers , which occurs following parp-1 automodification , covers the molecular weight region corresponding to the 180 kda of the modified ctcf ( 21 ) .
parp-1 poly(adp - ribosyl)ation increased with ctcf overexpression time with a maximum reached at 48 h. real time pcr and western blot analyses showed that this increase was not correlated with greater parp-1 and parp-2 mrna levels or with their higher nuclear levels ( fig .
1 , a and b ) . to directly test for the effect of ctcf on parp-1 activity , we used immunometric and radiometric methods .
recombinant parp-1 was incubated in the presence or absence of different amounts of recombinant bvctcf under conditions that are typically adopted to induce parp-1 automodification . the immunometric parp-1 activity assay ( fig .
1c ) was carried out in the presence of human recombinant parp-1 and dnase i - activated dna without ( lane 1 ) or with ( lane 2 ) pj-34 ( 20 m ) , a competitive inhibitor of parp activity .
dnase i - activated dna is dna on which some nicks are present , this condition being necessary to induce the poly(adp - ribosyl)ation of the nick - sensor parp-1 .
1c , lanes 46 ) or in the absence ( lanes 79 ) of pj-34 , plus addition of increasing amounts of recombinant ctcf ( ctcf / parp-1 molar ratio from 0.05 to 0.2 ) .
finally , parp activity was assayed following addition of recombinant ctcf ( at the same ctcf / parp-1 molar ratio ) but in absence of  activated  dna ( 1012 ) . fig .
1c , lane 3 , reports the reaction carried out in the absence of parp-1 , indicating the absence of parp activity in bvctcf preparation .
as shown , increasing the molar ratio of ctcf / parp-1 increased the level of poly(adp - ribosyl)ation of parp-1 .
it was surprising that ctcf acted as an activator of parp-1 modification per se , being able to induce poly(adp - ribosyl)ation of parp-1 even in the absence of nicked dna , which is required to allow parp-1 activity .
the presence of ctcf reduces the effect of the competitive inhibitor pj-34 , meaning that there may be conformational change of parp-1 .
in fact , poly(adp - ribosyl)ation of parp-1 was not completely inhibited by a pj-34 concentration of 20 m .
the molar ratio ctct / parp-1 of 0.4 increases parp-1 activity in presence of activated dna by about 15-fold .
this assay confirms the finding that ctcf is capable per se of inducing poly(adp - ribosyl)ation of parp-1 even in absence of activated dna .
in fact , the presence of ctcf at 0.4 pmol allows 8.5-fold increase in the modification of parp-1 .
co - immunoprecipitation and pulldown experiments have shown that ctcf and parp-1 directly interact both in vivo and in vitro .
1 , e and f ) .   figure 2.in vivo and in vitro experiments addressing the effect of ctcf overexpression on dna methyltransferase activity .
a , nuclear lysates from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci were analyzed by sds - page and western blot using antibodies against ctcf , dnmt1 , and against sp1 as endogenous control .
b , real time rt - pcr for dnmt1 performed on rna samples from cells at 24 , 48 , and 72 h post - transfection with either pci ( white bar ) or pci - ctcf ( gray bar ) vectors using hprt1 mrna as endogenous control .
of the arithmetic ratio of dnmt1 mrna level to hprt1 mrna level within each sample calculated from a minimum of three experiments performed in duplicate .
c , endogenous dna methyltransferase activity of nuclear extract from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf ( gray bar ) and pci ( white bar ) vectors .
d , in vitro activity assay of recombinant dnmt1 in the presence of increasing amounts of bvctcf . data report percentage of dna methyltransferase 1 activity following addition of bvctcf (  ) , in vitro poly(adp - ribosyl)ated parp-1 (  ) , parp-1 at a parp-1/dnmt1 molar ratio of 1 (  ) , and ctcf elution buffer ( 224 ) .
normal igg were used as negative control .   in vivo and in vitro experiments addressing the effect of ctcf overexpression on dna methyltransferase activity .
a , nuclear lysates from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci were analyzed by sds - page and western blot using antibodies against ctcf , dnmt1 , and against sp1 as endogenous control .
b , real time rt - pcr for dnmt1 performed on rna samples from cells at 24 , 48 , and 72 h post - transfection with either pci ( white bar ) or pci - ctcf ( gray bar ) vectors using hprt1 mrna as endogenous control .
data are reported as mean  s.e . of the arithmetic ratio of dnmt1 mrna level to hprt1 mrna level within each sample calculated from a minimum of three experiments performed in duplicate .
c , endogenous dna methyltransferase activity of nuclear extract from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf ( gray bar ) and pci ( white bar ) vectors .
d , in vitro activity assay of recombinant dnmt1 in the presence of increasing amounts of bvctcf . data report percentage of dna methyltransferase 1 activity following addition of bvctcf (  ) , in vitro poly(adp - ribosyl)ated parp-1 (  ) , parp-1 at a parp-1/dnmt1 molar ratio of 1 (  ) , and ctcf elution buffer ( 224 ) . data reported in b 
a , methyl - accepting ability assay was carried out on genomic dna purified from cells transfected for different times ( 24 , 48 , and 72 h ) with pci - ctcf ( gray bar ) or pci ( white bar ) vectors .
dna obtained from cells treated with 5-aza was used as positive control for genome hypomethylation ( black bar ) .
b and c , southern blot against minor satellite ( b ) and b1 ( c ) dna repeats performed on genomic dna purified from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci and digested with hpaii or mspi restriction enzymes .
dna obtained from cells treated with 5-aza was used as positive control for genome hypomethylation .  
a , methyl - accepting ability assay was carried out on genomic dna purified from cells transfected for different times ( 24 , 48 , and 72 h ) with pci - ctcf ( gray bar ) or pci ( white bar ) vectors .
dna obtained from cells treated with 5-aza was used as positive control for genome hypomethylation ( black bar ) .
b and c , southern blot against minor satellite ( b ) and b1 ( c ) dna repeats performed on genomic dna purified from cultures at 24 , 48 , and 72 h post - transfection with pci - ctcf and pci and digested with hpaii or mspi restriction enzymes .
dna obtained from cells treated with 5-aza was used as positive control for genome hypomethylation . our knowledge that dna methylation and poly(adp - ribosyl)ation are connected prompted us to further investigate the scenario where the ectopic overexpression of ctcf induced a burst of pars and poly(adp - ribosyl)ation of parp-1 , which remained high in time .
we expected that this condition would induce permanent inhibition of dnmt1 and consequently genomic dna hypomethylation .
we thus tested whether the increased amount of modified parp-1 influenced the endogenous dna methyltransferase activity .
the activity assay , carried out on nuclear extracts obtained from cells ectopically overexpressing ctcf and the respective control cells , showed that dna methyltransferase activity decreased by about 6570% ( fig . 2c ) .
2a ) excluded that inhibition of enzymatic activity was dependent on down - regulation of dnmt1 levels .
the possibility that ctcf per se was acting as an inhibitor of dnmt1 activity was disproved by in vitro assays ; parallel experiments carried out with in vitro poly(adp - ribosyl)ated parp-1 confirmed that poly(adp - ribosyl)ated parp-1 is responsible for inhibitory effect on dnmt1 ( 7 ) ( fig .
co - immunoprecipitation experiments , carried out on nuclear extracts from l929 mouse fibroblasts , indicate that parp-1 and dnmt1 are associated in vivo ( fig .
research was then focused on dna methylation pattern and the possibility that inhibition of dnmt1 activity leads to dna hypomethylation .
3a reports data from methyl - accepting ability assays , carried out on dna purified from cells overexpressing ctcf and control cells in the presence of labeled s - adenosylmethionine and human recombinant dnmt1 .
ctcf overexpression introduced hypomethylation on genomic dnas , and as a result , its methyl - accepting ability increased 3-fold at 24 h from transfection .
the integrity and the amounts of purified genomic dna were evaluated by agarose gel electrophoresis ( data not shown ) .
southern blot analyses were performed on the same dna samples to localize the new hypomethylated sequences .
3 , b and c , reports experiments in which genomic dna , previously digested with methylation - sensitive hpaii restriction enzyme and with its insensitive isoschizomer mspi , was analyzed , after electrophoretic separation of dna fragments , by using 3-digoxigenin - labeled single strand synthetic oligonucleotides as probes . as can be seen , both centromeric and b1 repetitive dna sequences were hypomethylated . to verify how specific the parp-1 poly(adp - ribosyl)ation is in affecting dna methylation activity , we performed experiments of ctcf overexpression in parp-1 mouse cells .
ectopic overexpression of ctcf in parp-1 cells did not reduce the dna methyltransferase activity , dnmt1 level , and the cell growth significantly ( fig .
4 , a  c and supplemental experimental procedures ) , although it induces a moderate increase of pars because of an enhanced activity of parp-2 and/or of other members of parp family ( fig .
figure 4.effect of ctcf overexpression in parp-1 cells on pars level , cell survival , dnmt1 expression , and dna methyltransferase activity .
a , nuclear lysates from l929 and a1 ( parp-1 ) cells at 72 h post - transfection with pci and pci - ctcf vectors were analyzed by sds - page and western blotting , using antibodies against par and against the indicated proteins .
b , survival of l929 and a1 cells after 72 h post - transfection with pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
c , endogenous dnmt activity of nuclear lysates from l929 and a1 cells at 72 h post - transfection with pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
effect of ctcf overexpression in parp-1 cells on pars level , cell survival , dnmt1 expression , and dna methyltransferase activity .
a , nuclear lysates from l929 and a1 ( parp-1 ) cells at 72 h post - transfection with pci and pci - ctcf vectors were analyzed by sds - page and western blotting , using antibodies against par and against the indicated proteins .
b , survival of l929 and a1 cells after 72 h post - transfection with pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
c , endogenous dnmt activity of nuclear lysates from l929 and a1 cells at 72 h post - transfection with pci ( white bar ) and pci - ctcf ( gray bar ) vectors .
this research emphasizes the importance of proper dynamics between poly(adp - ribose ) polymerase and poly(adp - ribose ) glycohydrolase ( parg ) activities so that cellular homeostasis is not affected by low or excessively high par levels . in physiological conditions ,
the active , poly(adp - ribosyl)ated parp-1 molecules constitute a small proportion of the total population of parp-1 molecules , whereas upon injury to the cell , the number of active parp-1 molecules increases causing an up to 500 times higher level of pars ( 28 ) .
in this environment parp-1 plays an important role as it is responsible for about 90% of par production ( 29 ) .
however , the basal level of polymers is rapidly restored thanks to the intervention of parg ( 3032 ) , which shuttles from the cytoplasm to the nucleus ( 33 , 34 ) , digesting the excess of nuclear pars in a few minutes ( 28 , 35 ) .   figure 5.a possible model describing the sequence of events occurring in cells following ctcf ectopic overexpression .
ctcf activates ( gray arrow ) parp-1 inducing its automodification and poly(adp - ribosyl)ation of ctcf and of other nuclear proteins yet to be identified .
modified parp-1 , binding noncovalently the dnmt1 , inhibits its enzymatic activity , and as a consequence , the genomic dna becomes widely hypomethylated .   a possible model describing the sequence of events occurring in cells following ctcf ectopic overexpression
. ctcf activates ( gray arrow ) parp-1 inducing its automodification and poly(adp - ribosyl)ation of ctcf and of other nuclear proteins yet to be identified .
the persistence of pars in the nuclear environment induces  stable  inhibition of dnmt1 activity .
modified parp-1 , binding noncovalently the dnmt1 , inhibits its enzymatic activity , and as a consequence , the genomic dna becomes widely hypomethylated . over the years
, it has been shown that the decrease and increase of par level can both threaten the normal cellular physiology .
treatment of cells with competitive inhibitors of parp activity has demonstrated that delay in dna damage repair and genomic instability occur when nuclear levels of pars are low ( 36 ) .
research on cells knocked out for parp-1 ( 37 ) or parp-2 ( 38 ) , the two best - known members of the parp family ( 39 ) , has indicated that both enzymes are involved in the mechanism of dna damage repair : knocking out of either enzyme is not lethal , whereas the cell can not survive the double knock - outs ( 38 ) .
likewise harmful is the accumulation of pars , which is even perceived as a signal for cell death ( 4042 ) .
cell death , observed when par level is high , has long been attributed to nad depletion and thus to energy insufficiency ( 43 ) . without excluding that more than one event leads to cellular death
, it has been shown that pars by themselves cause cell mortality ( 44 ) .
cytoplasmic long and branched polymers ( 41 ) , interacting with mitochondrial membrane stimulate the release of the pro - apoptotic factor aif , an apoptotic signal downstream of parp activation ( 42 , 45 , 46 ) . in the above - mentioned research attention was focused on what happens to the cell when high levels of pars are present in the cytosol .
we have focused our attention on events that occur in the nucleus . based on our experimental data
the starting point is when the increased nuclear level of ctcf induces a higher level of nuclear pars .
it is surprising that at all observation times , 24 , 48 , and 72 h following ectopic transfection of ctcf , the nuclear level of pars remained high not depending on an increased parp-1 expression , even though the par level returns to basal level in a few minutes following stress ( 28 ) . in vitro experiments
this activation happens even in absence of activated dna , and this is different from yy1 ( 47 ) and vp1 ( 48 ) .
co - immunoprecipitation and pulldown experiments confirmed interaction between ctcf and parp-1 both in vivo and in vitro .
our previous data show that both free pars and pars present on modified parp-1 , through a noncovalent interaction with dnmt1 , inhibit its enzymatic activity . here
we show that dna methyltransferase activity is decreased by about 70% in nuclear extract in cells overexpressing ctcf versus cells transfected with empty vector .
a more thorough examination of dnmt1 has shown that the inhibition is not because of a decreased level of dnmt1 expression .
an in vitro approach allowed us to exclude direct inhibition of dnmt1 activity by ctcf , in line with our previous observation that pars present on modified parp-1 inhibit dnmt1 activity ( 7 ) .
the persistence of a high par level affects the dna methylation machinery ; dnmt1 activity is inhibited , leading to wide hypomethylation of the genome , involving both centromeric and b1 repeats .
ectopic overexpression of ctcf in parp-1 cells demonstrates a specific functional relationship between ctcf and parp-1 .
this finding underscores the importance of a rapid reversal of parp-1 automodification because is affects dna methylation patterns considered to be part of the epigenetic code .
dna hypomethylation could decondense chromatin structure ( 49 , 50 ) , cause genomic instability ( 51 ) , direct cells toward apoptosis ( 52 ) , and cancer ( 53 , 54 ) .
the unusual cellular environment , in which a large amount of adp - ribose polymers are present for several days , mimics the situation in parg knock - outs and provides a model for the study of damage because of excess of adp - ribose polymers in cells .
our data suggest that deregulation of dna methylation pattern is one of the molecular mechanisms causing lethality in parg knock - out mice ( 40 ) .
the finding that high par levels induce hypomethylation is intriguing , because in tumor cells the hypomethylation of repeat - rich heterochromatin contributes to genomic instability , through increased mitotic recombination events ( 51 ) .
the introduction of new methyl groups onto dna or the diffuse hypomethylation in cancer cells ( 54 , 56 ) could occur through deregulation of parp or parg activities .
this demethylation is a passive mechanism ( 55 ) that does not depend on decreased expression of dnmt1 but on the absence of an active dnmt1 . in the cellular scenario
dependent on ectopic overexpression of ctcf , the cytotoxicity is accounted for by a decreased level of intracellular nad ( 43 ) , whereas the increased number of apoptotic cells can be explained by recent data showing that the higher adp - ribose polymer production ( 57 ) and dna hypomethylation ( 52 ) induce apoptosis .
the connection between dna methylation and poly(adp - ribosyl)ation inferred from our previous studies ( 16 ) is strengthened by our new findings showing that activation of parp-1 , dependent on ctcf , affects dna methylation machinery .
it is clear that , because of the complexity of pathways that occur in cells , despite evidence showing the connection between poly - adp - ribosylated parp-1 and dnmt1 , other additional and/or alternative mechanism(s ) can not be excluded .
based on our extensive research on poly(adp - ribosyl)ation and dna methylation , we think that the control of dna methylation pattern is among the multiple housekeeping roles of parp-1 ; this role could be played by the inhibitory effect exerted by pars on the dnmt1 ( 7 ) .
the balance of pars constitutively present in cells may be extremely important ; their decrease causes anomalous dna hypermethylation ( 16 ) , whereas their increase leads to widespread dna hypomethylation .
further research will be carried out to define in depth the role played by ctcf in the cross - talk between poly(adp - ribosyl)ation and dna methylation and to establish whether and how the normal functions of parg are affected in this new cellular scenario .
we will assess whether ctcf represses parg expression , inhibits its enzymatic activity , or impairs its physiological shuttling from the cytoplasm to the nucleus .
 our previous data have shown that in l929 mouse fibroblasts the control of methylation pattern depends in part on poly(adp - ribosyl)ation and that adp - ribose polymers ( pars ) , both present on poly(adp - ribosyl)ated parp-1 and/or protein - free , have an inhibitory effect on dnmt1 activity . here 
 we show that transient ectopic overexpression of ccctc - binding factor ( ctcf ) induces par accumulation , parp-1 , and ctcf poly(adp - ribosyl)ation in the same mouse fibroblasts . 
 the persistence in time of a high par level affects the dna methylation machinery ; the dna methyltransferase activity is inhibited with consequences for the methylation state of genome , which becomes diffusely hypomethylated affecting centromeric minor satellite and b1 dna repeats . in vitro data show that ctcf is able to activate parp-1 automodification even in the absence of nicked dna . 
 our new finding that ctcf is able per se to activate parp-1 automodification in vitro is of great interest as so far a burst of poly(adp - ribosyl)ated parp-1 has generally been found following introduction of dna strand breaks . 
 ctcf is unable to inhibit dnmt1 activity , whereas poly(adp - ribosyl)ated parp-1 plays this inhibitory role . 
 these data suggest that ctcf is involved in the cross - talk between poly(adp - ribosyl)ation and dna methylation and underscore the importance of a rapid reversal of parp activity , as dna methylation pattern is responsible for an important epigenetic code . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material