tight junctions , the most apical component of the epithelial junctional complex , recruit signalling proteins such as protein kinases , rho gtpase regulators and transcription factors that can localize to junctions as well as to nuclei ( aijaz et al , 2006 ) . however , there is little understanding of how the different junction - associated signalling molecules are interconnected and coordinate cellular responses . zonab ( zonula occludens 1 ( zo-1)-associated nucleic - acid- binding protein)/dbpa ( dna - binding protein a ) is a y - box transcription factor that is recruited to tight junctions by binding to the src homology 3 domain of zo-1 ( balda & matter , 2000 ) . zonab regulates epithelial proliferation by restricting g1s phase progression ( balda et al , 2003 ) . its transcriptional activity is important for this regulation because one of the target genes of zonab is cyclin d1 , which is a key cell - cycle regulator ( sourisseau et al , 2006 ) . as zo-1 binding to zonab results in inhibition , tight junction formation suppresses this proliferation - promoting pathway . the reported deregulated expression of zo-1 and zonab in different tumours might , therefore , contribute to the transformed phenotype ( hoover et al , 1998 ; martin et al , 2004 ; yasen et al , 2005 ) . binding of activated rala also inhibits transcriptional activation of zonab ( frankel et al , 2005 ) . by contrast , the mechanisms that mediate zonab activation are poorly understood : apg-2 , a heat - shock protein , can stimulate zonab activation indirectly by binding to zo-1 ( tsapara et al , 2006 ) ; but mechanisms that activate zonab directly are not known . another tight - junction - associated signalling component that promotes g1s phase progression is guanine nucleotide exchange factor ( gef)-h1/lfc ( benais - pont et al , 2003 ; aijaz et al , 2005 ) . gef - h1 is an exchange factor for rhoa , and it has been proposed that its deregulation contributes to carcinogenesis ( birkenfeld et al , 2008 ) . in epithelial cells , gef - h1 activates rhoa signalling , regulating paracellular permeability , junctional dynamics and proliferation ( benais - pont et al , 2003 ; aijaz et al , 2005 ; ivanov et al , 2005 ) . rhoa signalling regulates g1s phase transition , and one of the crucial target genes of rho signalling during this cell - cycle step is the cyclin d1 gene ( coleman et al , 2004 ; ridley , 2004 ) . however , if and how gef - h1 participates in the regulation of gene expression is not known . cyclin d1 is upregulated during g1 phase and stimulates g1s phase transition by binding and activating cyclin - dependent kinases 4/6 , which contributes to cyclin e  cdk2 activation and inactivation of the cell - cycle inhibitory function of retinoblastoma protein ( prb ) by hyperphosphorylation ( fu et al , 2004 ) . cyclin d1 also has a role during other cell - cycle phases that require careful regulation of localization and degradation by phosphorylation and the ubiquitin / proteosome system ( sherr , 2002 ; gladden & diehl , 2005 ) . here , we show that two tight - junction - associated signalling proteins , zonab and gef - h1 , form a complex and that activation of the transcriptional activity of zonab is regulated by rho . our results indicate that zonab and gef - h1 form a signalling module that links a rho activator to a downstream signalling effector and mediates rho - regulated cyclin d1 expression . zonab and rho gtpase signalling regulate g1s phase progression and cyclin d1 expression ( balda et al , 2003 ; coleman et al , 2004 ; ridley , 2004 ; sourisseau et al , 2006 ) . as gef - h1 , a gef for rhoa , regulates epithelial cell proliferation ( aijaz et al , 2005 ) , we first tested whether gef - h1 is also required for cyclin d1 expression . our experimental system used madin  darby canine kidney ( mdck ) cells , which allow tetracycline - regulated conditional depletion of zonab and gef - h1 ( fig 1a ; aijaz et al , 2005 ; sourisseau et al , 2006 ) . to monitor the expression levels during g1s phase progression , cells were arrested in low serum and then stimulated by adding back serum , which results in efficient re - entry of mdck cells into the cell cycle and effective progression into s phase in 24 h ( balda et al , 2003 ) . depletion of both zonab and gef - h1 resulted in a strong reduction in cyclin d1 expression in cells that were stimulated with serum after synchronization in low serum ( fig 1 ) . overexpression of gef - h1 , but not zonab , was sufficient to stimulate increased cyclin d1 levels in arrested cells ( 0 h ) , indicating that zonab requires serum stimulation for activity . in confluent cells , expression of cyclin d1 was low in all cell lines ( supplementary fig s1 online ) . these data show that gef - h1 regulates cyclin d1 expression in proliferating cells and , similarly to zonab , is required for serum - induced cyclin d1 upregulation . gef - h1 and zonab associate with tight junctions but are also found at other subcellular locations in epithelial cells ( balda & matter , 2000 ; benais - pont et al , 2003 ) . by immunofluorescence , the two proteins co - localize with zo-1 at intercellular junctions , indicating that the two proteins might exist in a complex ( supplementary fig s2 online ) . therefore , we tested whether gef - h1 and zonab form a complex in mdck cells by using immunoprecipitation and by monitoring co - precipitation through immunoblotting . fig 2a shows that gef - h1 was detected in zonab but not in control immunoprecipitates . similarly , zonab was detected in precipitates with gef - h1-specific antibodies , although co - precipitation was less efficient . co - precipitation of gef - h1 was seen in confluent , but not in proliferating , subconfluent cells that had been cultured for 2 days , indicating that the complex does not exist constitutively ( fig 2b ) . similarly , gef - h1 is less junctional in low confluent than in confluent cells ( supplementary fig s3 online ) . these data indicate that gef - h1 and zonab can form a complex in epithelial cells and that complex formation depends on cell confluence . glutathione s - transferase ( gst ) fusion proteins containing different domains of gef - h1 were then used to map the binding site for zonab ( fig 2c ; aijaz et al , 2005 ) . pull - down experiments with mdck cell extracts revealed that the c1 domain is sufficient to mediate the interaction with zonab ( fig 2d ) . zonab is expressed in two isoforms , zonab - a and -b , and both could be pulled down efficiently with the c1 fusion protein from extracts of cos-7 cells transfected with vesicular stomatitis virus ( vsv)-tagged zonab isoforms ( fig 2e ) . as both zonab - a and -b co - immunoprecipitated with gef - h1 , both seem to be able to form a complex with the exchange factor . we then used recombinant his - tagged zonab to determine whether the interaction could be reconstituted in vitro . fig 2f , g shows that recombinant zonab was precipitated by glutathione s - transferase ( gst)-c1 but not gst alone , indicating that the c1 domain of gef - h1 can bind directly to zonab . zonab is thus far the only gef - h1-interacting protein known to interact with gst - c1 , as other proteins require different or further domains ( for example , microtubule - affinity regulating kinase / partition - defective-1 ( mark / par1 ) needs an additional intervening domain ; supplementary fig s4 online ) . as overexpression of gef - h1 is sufficient to stimulate rhoa activation ( birkenfeld et al , 2008 ) , we first tested whether overexpression stimulates nuclear translocation of zonab . fig 3 shows that overexpression of gef - h1 did result in nuclear accumulation of zonab . the remaining cytoplasmic , dotty staining often seemed to follow the cytoplasmic gef - h1 staining . gef - h1 was no longer detected at junctions but , as indicated in existing reports ( birkenfeld et al , 2008 ) , was cytosolic and partially associated with microtubules . mark / par1 , another gef - h1 interaction partner in mdck cells that was originally identified in a proteomics screen ( brajenovic et al , 2004 ) , also dissociated from cell  cell contacts on gef - h1 overexpression , but remained cytoplasmic and partially co - localized with gef - h1 ( fig 3c and supplementary fig s4 online ) . induction of gef - h1 expression induced nuclear translocation of zonab without affecting the expression levels of zonab and zo-1 ( supplementary fig s5 online ) . we next analysed whether rho signalling and gef - h1 regulate the transcriptional activity of zonab . one driving firefly luciferase expression and the other driving renilla luciferase expression  that differ only by the presence of a zonab binding site in one of them ( frankel et al , 2005 ) . zonab functions as a repressor of the promoter with a functional zonab binding site ( balda & matter , 2000 ) . expression of dominant - negative rhoa ( t19n - rhoa ) stimulated the zonab - responsive promoter , reflecting zonab inhibition ( fig 4a ) . accordingly , a constitutively active form ( v14-rhoa ) increased zonab activity , and incubation with membrane - permeable c3 transferase  which inactivates rhoa , b and c  reduced zonab activity . as zonab is active in low - density , proliferating cells , but not in high - density cells , promoter stimulation reflecting zonab inactivation was observed only in low - density cultures . overexpression of gef - h1 also repressed the promoter , indicating zonab activation ( fig 4b ) . stimulation did not require the c1 domain , which binds to zonab , but overexpression of an active exchange factor domain was sufficient , as expected from the constitutively active rhoa mutant result . this indicates that a direct interaction is not required for regulation when active rhoa levels are very high ( that is , expression of constitutively active gef or rhoa ) . however , depletion of gef - h1 inhibited zonab activity in low - density cells to a similar extent as did zonab depletion , indicating that gef - h1 is a functionally relevant zonab activator ( fig 4c ) . expression of the zonab - interacting c1 domain inhibited the transcription factor in low - density cells , indicating that it functions as a dominant mutant . as the c1 domain co - localized with endogenous zonab but did not affect nuclear accumulation , inhibition might have been due to its prevention of another interaction required for transcription ( supplementary fig s6 online ) . in addition , expression of the c1 domain did not affect the distribution of gef - h1 ( supplementary fig s7 online ) . zonab rna interference ( rnai ) was then combined with the constitutively active gef - h1 mutant to confirm the involvement of zonab in the changes observed in promoter activation . fig 4d shows that in high - density cells , in which zonab is not active , constitutively active gef - h1 only repressed the promoter in the presence of zonab ( with or without co - transfection of a control rnai plasmid ) but not when the transcription factor was depleted . this confirms the requirement of zonab for repression of the promoter by gef - h1 . zonab and gef - h1 regulate expression of cyclin d1 ( fig 1 ) . to determine whether this involves changes in cyclin d1 promoter activity , we used a firefly luciferase reporter construct containing a full - length cyclin d1 promoter with a zonab binding site at position 530 ( albanese et al , 1995 ; sourisseau et al , 2006 ) . however , when the zonab rnai plasmids were co - transfected , stimulation was strongly reduced . zonab and rho gtpase signalling regulate g1s phase progression and cyclin d1 expression ( balda et al , 2003 ; coleman et al , 2004 ; ridley , 2004 ; sourisseau et al , 2006 ) . as gef - h1 , a gef for rhoa , regulates epithelial cell proliferation ( aijaz et al , 2005 ) , we first tested whether gef - h1 is also required for cyclin d1 expression . our experimental system used madin  darby canine kidney ( mdck ) cells , which allow tetracycline - regulated conditional depletion of zonab and gef - h1 ( fig 1a ; aijaz et al , 2005 ; sourisseau et al , 2006 ) . to monitor the expression levels during g1s phase progression , cells were arrested in low serum and then stimulated by adding back serum , which results in efficient re - entry of mdck cells into the cell cycle and effective progression into s phase in 24 h ( balda et al , 2003 ) . depletion of both zonab and gef - h1 resulted in a strong reduction in cyclin d1 expression in cells that were stimulated with serum after synchronization in low serum ( fig 1 ) . overexpression of gef - h1 , but not zonab , was sufficient to stimulate increased cyclin d1 levels in arrested cells ( 0 h ) , indicating that zonab requires serum stimulation for activity . in confluent cells , expression of cyclin d1 was low in all cell lines ( supplementary fig s1 online ) . these data show that gef - h1 regulates cyclin d1 expression in proliferating cells and , similarly to zonab , is required for serum - induced cyclin d1 upregulation . gef - h1 and zonab associate with tight junctions but are also found at other subcellular locations in epithelial cells ( balda & matter , 2000 ; benais - pont et al , 2003 ) . by immunofluorescence , the two proteins co - localize with zo-1 at intercellular junctions , indicating that the two proteins might exist in a complex ( supplementary fig s2 online ) . therefore , we tested whether gef - h1 and zonab form a complex in mdck cells by using immunoprecipitation and by monitoring co - precipitation through immunoblotting . fig 2a shows that gef - h1 was detected in zonab but not in control immunoprecipitates . similarly , zonab was detected in precipitates with gef - h1-specific antibodies , although co - precipitation was less efficient . co - precipitation of gef - h1 was seen in confluent , but not in proliferating , subconfluent cells that had been cultured for 2 days , indicating that the complex does not exist constitutively ( fig 2b ) . similarly , gef - h1 is less junctional in low confluent than in confluent cells ( supplementary fig s3 online ) . these data indicate that gef - h1 and zonab can form a complex in epithelial cells and that complex formation depends on cell confluence . glutathione s - transferase ( gst ) fusion proteins containing different domains of gef - h1 were then used to map the binding site for zonab ( fig 2c ; aijaz et al , 2005 ) . pull - down experiments with mdck cell extracts revealed that the c1 domain is sufficient to mediate the interaction with zonab ( fig 2d ) . zonab is expressed in two isoforms , zonab - a and -b , and both could be pulled down efficiently with the c1 fusion protein from extracts of cos-7 cells transfected with vesicular stomatitis virus ( vsv)-tagged zonab isoforms ( fig 2e ) . as both zonab - a and -b co - immunoprecipitated with gef - h1 , both seem to be able to form a complex with the exchange factor . we then used recombinant his - tagged zonab to determine whether the interaction could be reconstituted in vitro . fig 2f , g shows that recombinant zonab was precipitated by glutathione s - transferase ( gst)-c1 but not gst alone , indicating that the c1 domain of gef - h1 can bind directly to zonab . zonab is thus far the only gef - h1-interacting protein known to interact with gst - c1 , as other proteins require different or further domains ( for example , microtubule - affinity regulating kinase / partition - defective-1 ( mark / par1 ) needs an additional intervening domain ; supplementary fig s4 online ) . as overexpression of gef - h1 is sufficient to stimulate rhoa activation ( birkenfeld et al , 2008 ) , we first tested whether overexpression stimulates nuclear translocation of zonab . fig 3 shows that overexpression of gef - h1 did result in nuclear accumulation of zonab . the remaining cytoplasmic , dotty staining often seemed to follow the cytoplasmic gef - h1 staining . gef - h1 was no longer detected at junctions but , as indicated in existing reports ( birkenfeld et al , 2008 ) , was cytosolic and partially associated with microtubules . mark / par1 , another gef - h1 interaction partner in mdck cells that was originally identified in a proteomics screen ( brajenovic et al , 2004 ) , also dissociated from cell  cell contacts on gef - h1 overexpression , but remained cytoplasmic and partially co - localized with gef - h1 ( fig 3c and supplementary fig s4 online ) . induction of gef - h1 expression induced nuclear translocation of zonab without affecting the expression levels of zonab and zo-1 ( supplementary fig s5 online ) . we next analysed whether rho signalling and gef - h1 regulate the transcriptional activity of zonab . one driving firefly luciferase expression and the other driving renilla luciferase expression  that differ only by the presence of a zonab binding site in one of them ( frankel et al , 2005 ) . zonab functions as a repressor of the promoter with a functional zonab binding site ( balda & matter , 2000 ) . expression of dominant - negative rhoa ( t19n - rhoa ) stimulated the zonab - responsive promoter , reflecting zonab inhibition ( fig 4a ) . accordingly , a constitutively active form ( v14-rhoa ) increased zonab activity , and incubation with membrane - permeable c3 transferase  which inactivates rhoa , b and c  reduced zonab activity . as zonab is active in low - density , proliferating cells , but not in high - density cells , promoter stimulation reflecting zonab inactivation was observed only in low - density cultures . overexpression of gef - h1 also repressed the promoter , indicating zonab activation ( fig 4b ) . stimulation did not require the c1 domain , which binds to zonab , but overexpression of an active exchange factor domain was sufficient , as expected from the constitutively active rhoa mutant result . this indicates that a direct interaction is not required for regulation when active rhoa levels are very high ( that is , expression of constitutively active gef or rhoa ) . however , depletion of gef - h1 inhibited zonab activity in low - density cells to a similar extent as did zonab depletion , indicating that gef - h1 is a functionally relevant zonab activator ( fig 4c ) . expression of the zonab - interacting c1 domain inhibited the transcription factor in low - density cells , indicating that it functions as a dominant mutant . as the c1 domain co - localized with endogenous zonab but did not affect nuclear accumulation , inhibition might have been due to its prevention of another interaction required for transcription ( supplementary fig s6 online ) . in addition , expression of the c1 domain did not affect the distribution of gef - h1 ( supplementary fig s7 online ) . zonab rna interference ( rnai ) was then combined with the constitutively active gef - h1 mutant to confirm the involvement of zonab in the changes observed in promoter activation . fig 4d shows that in high - density cells , in which zonab is not active , constitutively active gef - h1 only repressed the promoter in the presence of zonab ( with or without co - transfection of a control rnai plasmid ) but not when the transcription factor was depleted . this confirms the requirement of zonab for repression of the promoter by gef - h1 . zonab and gef - h1 regulate expression of cyclin d1 ( fig 1 ) . to determine whether this involves changes in cyclin d1 promoter activity , we used a firefly luciferase reporter construct containing a full - length cyclin d1 promoter with a zonab binding site at position 530 ( albanese et al , 1995 ; sourisseau et al , 2006 ) . however , when the zonab rnai plasmids were co - transfected , stimulation was strongly reduced . epithelial junctions recruit various signalling proteins , and several of them are thought to regulate proliferation and differentiation . unravelling the biochemical and functional networks that these proteins form is therefore important in order to understand the signalling mechanisms they are involved in . the data presented here show that gef - h1 and zonab form a signalling module that mediates rho - stimulated transcription . the transcriptional activity of zonab is rho - regulated as both dominant - negative rhoa and c3 transferase inhibit its activity . how rho activation stimulates zonab is not yet clear . inhibition of rho signalling by inhibition of rho - associated kinase ( rock ) and myosin , or transfection of dominant - negative dia , therefore , it might be that a yet - to - be - identified rho effector stimulates zonab activation . alternatively , it is possible that inactive gef - h1 and zonab form a complex in the absence of a stimulus that localizes at tight junctions and is thus primarily observed in confluent cultures . gef - h1-mediated rho activation then leads to a dissociation of the complex and , hence , activation of zonab . given the absence of gef - h1 from junctions in overexpressing cells , rho activation might also lead to an inactivation of the mechanisms that recruit gef - h1 complexes to the junction . gef - h1 has already been linked to rho / rock / myosin - regulated junctional dynamics and dissociation ( aijaz et al , 2006 ; samarin et al , 2007 ) . it thus seems that gef - h1 functions as a junction - associated rho signalling hub that activates rho and , subsequently , different rho effector pathways that then drive junctional rearrangements as well as transcriptional responses . recently , gef - h1 has also been found to be activated in response to inflammatory stimuli ( fukazawa et al , 2008 ; kakiashvili et al , 2009 ) , which is also observed in mdck cells ( supplementary fig s8 online ) . however , at least one such stimulator , tnf , does not stimulate the transcriptional activity of zonab ( data not shown ) , suggesting that additional signalling mechanisms act on zonab to modulate its activation . one possibility is that rala binding inhibits tnf-induced zonab activation : rala binds to and inhibits zonab and is activated in response to tnf ( sugihara et al , 2002 ; frankel et al , 2005 ) . perhaps the best studied one is the regulation of serum response factor , a transcription factor that is activated by a co - activator in which nuclear accumulation is regulated by rho gtpases through actin polymerization ( miralles et al , 2003 ) . another type of rho regulation is represented by nf-b , a transcription factor that is modulated by rho gtpases and rho kinases either directly or indirectly by phosphorylation of upstream regulators ( perkins , 2007 ) . to our knowledge , zonab is the first case of a rho - regulated transcription factor that associates with a rho gef . thus , gef - h1 and zonab form a new type of signalling module that spatially links a rho activator to an effector pathway to transmit signals to the nucleus . cell lines , expression constructs , rnai and antibodies . mdck cells for tetracycline - regulated gef - h1 expression , and rnai of gef - h1 and zonab were described previously ( aijaz et al , 2005 ; sourisseau et al , 2006 ) . gef - h1 constructs fused to vsv and gst were described by aijaz et al ( 2005 ) and zonab expression and rnai plasmids by balda et al ( 2003 ) . the cdnas encoding the four ha - tagged marks were provided by gerard drewes ( cellzome ag , heidelberg , germany ) . fixation with methanol , and processing and analysis of cells for immunofluorescence were performed as described ( balda et al , 2003 ) . immunoprecipitations , immunoblotting , fusion protein production and pull - down assays were described by aijaz et al ( 2005 ) . see supplementary information online for sources of antibodies used and details of protein analysis , including cell culture mdck cells were plated into 96-well dishes at a low ( 2,000 cells / well ) or high ( 12,000 cells / well ) concentration the day before transfection . to measure zonab activity , two plasmids were co - transfected using the calcium phosphate method : one with a minimal promoter with a zonab binding site driving firefly luciferase expression , and the other with an identical promoter but with a mutant zonab binding site driving renilla luciferase expression ( balda & matter , 2000 ; frankel et al , 2005 ) . all expression and rnai plasmids that were co - transfected were mixed with the reporter plasmids before forming the precipitate ; total dna concentrations were kept constant with empty vectors . after 2 days , the two luciferases were measured sequentially using the dual luciferase assay kit ( promega corp , madison , wi , usa ) . the activity of the cyclin d1 promoter was assayed as described previously ( sourisseau et al , 2006 ) .
epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation . 
 little is known about how such proteins interact functionally and biochemically with each other . here , we focus on the y - box transcription factor zonab ( zonula occludens 1-associated nucleic - acid - binding protein)/dbpa ( dna - binding protein a ) and the rho gtpase activator guanine nucleotide exchange factor ( gef)-h1/lbc 's first cousin , which are two tight - junction - associated signalling proteins that regulate proliferation . 
 our data show that the two proteins interact and that zonab activity is rho - dependent . 
 overexpression of gef - h1 induces accumulation of zonab in the nucleus and activates transcription . 
 microtubule - affinity regulating kinase / partition - defective-1 , another type of gef - h1-associated signalling protein , remains in the cytoplasm and partially co - localizes with the exchange factor . 
 gef - h1 and zonab are required for expression of endogenous cyclin d1 , a crucial rhoa signalling target gene , and gef - h1-stimulated cyclin d1 promoter activity requires zonab . our data thus indicate that gef - h1 and zonab form a signalling module that mediates rho - regulated cyclin d1 promoter activation and expression .