a critical step in regulating many transcriptional responses is the import of transcription factors from the cytosol to the nucleus . many transcription factors are held outside the nucleus in a complex with cytosolic proteins or with membrane receptors , and translocate to the nucleus in response to various stimuli . alternatively , transcription factors may be inserted directly into the membrane , thereby preventing their access to the nucleus . a handful of such proteins has been shown to be released from membranes by a process known as regulated intramembrane proteolysis ( rip ) . this process is best understood for srebp-1 and srebp-2 , two basic leucine zipper ( bzip ) transcription factors that normally reside in the membrane of the endoplasmic reticulum and golgi apparatus . when cellular sterol levels dip , srebps are liberated from the membrane in a two - step mechanism involving the action of site-1 protease , a site - specific protease that cleaves the protein within the golgi lumen , followed by site-2 protease , an integral membrane protease , that cleaves a membrane - spanning helix . once liberated from the membrane , transport to the nucleus enables these transcription factors to initiate expression of genes involved in cholesterol uptake and biosynthesis . several more examples of membrane - tethered transcriptional regulators have recently been identified by biochemical means , notably atf6 , g13 , cadc , toxr , lzip ( luman ) , notch   and spt23 . all appear to undergo proteolytic cleavages to release a fragment that is targeted to dna or the nucleus , but may use different proteases . for example , atf6 uses the same proteolytic machinery , as do srebps , whereas notch is cleaved by different proteases . tumor necrosis factor ( tnf)-converting enzyme catalyzes the cleavage of the extracellular domain of notch , followed by presenilin / gamma - secretase - like activity to liberate the intracellular fragment . thus , the release of some membrane - bound nuclear proteins involves regulated cleavages in the lumenal or extracellular space , followed by a cleavage by an integral membrane protease to release an active fragment . using conventional biochemistry , the identification of transmembrane transcription factors ( tmtfs ) can be easily overlooked . for example , transcription factors are generally assumed to be soluble proteins and , consequently , membrane fractions are often discarded during purification . moreover , the nuclear form of the protein may be rapidly degraded and thus difficult to detect , as is the case for srebps . cell - fractionation studies of other transcription factors show smaller - molecular - weight forms of these proteins enriched in the nucleus , suggestive of a cleavage event . we thus investigated the prevalence of transmembrane transcription factors using computational tools to search for membrane - spanning proteins that contain conserved dna - binding domains . we used pfam   hidden markov models for 53 dna - binding domains ( see materials and methods ) to search all proteins in swiss - prot / trembl   and swisspfam protein databases . the 9,261 proteins identified by our search are presumed members of dna - binding protein families , and most are expected to be transcription factors . these proteins were then scored for the presence of one or more transmembrane segments using prediction programs phdhtm [ l8 ] , tmhmm , hmmtop   and psortii . only those proteins containing membrane - spanning helices predicted by at least three of the four programs were deemed significant in our analysis . by these stringent criteria , 76 proteins from 20 organisms and our analysis predicted a surprisingly large and diverse set of membrane - tethered dna - binding proteins . seventeen of the 53 dna - binding domains chosen for this analysis were represented in the final set of tmtfs . of these , the most abundant is the zf - c4 ( zinc - finger type c4 ) nuclear hormone receptor dna - binding domain , found in 14 proteins in caenorhabditis elegans and avian erythroblastosis virus . tmtfs in arabidopsis were the most diverse , and were associated with eight different dna - binding domains . all but two proteins have dna - binding domains that could be separated from the rest of the protein by a single hypothetical cleavage event , if singly predicted transmembrane segments are discounted ( figure 1 ) . dna - binding domains were also frequently juxtaposed to bipartite nuclear localization signals , suggesting that transmembrane and dna - binding domains in tmtfs are modular . thus , the overall topology of these proteins is consistent with other known tmtfs . c. elegans has an impressive 25 predicted tmtfs , suggesting that rip may be particularly important in the regulation of transcriptional responses in the worm . we deliberately used a stringent method to increase the likelihood of identifying only bona fide tmtfs and , as expected , most experimentally known tmtfs were detected by our analysis , including cadc , lzip , toxr   and all swiss - prot / trembl orthologs of srebp - l and srebp-2 . also found were several well - characterized proteins whose predicted membrane insertion had not been recognized . for example , the human doublesex - related protein dmrt2 , drosophila b - h2 ( barh2 ) protein , c. elegans unc-86 , and mouse oasis protein are predicted tmtfs . the transmembrane helix of atf6 was predicted by only two programs : psort and hmmtop . the immunoglobulin dna - binding domain ( tig ) of spt23 is found in both cell - surface proteins as well as transcription factors and was therefore excluded from the set of dna - binding domains . these results indicate that reducing the stringency of our prediction method will expand the number of predicted tmtfs . in some cases we found data in the literature to support our computational predictions . for example , cell - fractionation studies using antibodies directed at the carboxyl terminus of the chaperonin mtj1 showed that the full - length ( 62 kda ) protein exists in microsomes , whereas a smaller 42 kda form of the protein is found in the nucleus . the 42 kda species was hypothesized to represent a product of internal translation . because mtj1 contains putative myb dna - binding domains within the carboxy - terminal half of the protein , we re - examined the subcellular localization of carboxy - terminal - tagged mtj1 in cos-7 cells ( figure 2a ) . our results show clearly that full - length mtj1 is normally associated with the endoplasmic reticulum . in contrast , a truncated form mtj1 ( approximately 40 kda in size ) lacking the transmembrane segment accumulates in the nucleus . therefore , we propose that the 42 kda nuclear form of mtj1 observed in cells arises by cleavage of mtj1 from the membrane , rather than from aberrant translation of the mrna . dmrt2 , a human homolog of c. elegans mab-3 , was identified in our analysis as having a carboxy - terminal transmembrane segment ( figure 1 ) . dmrt2 has gained recent attention as a candidate gene for sex - reversal phenotypes in humans . to verify our prediction that dmrt2 is a membrane - tethered transcription factor , we examined the subcellular localization of full - length and truncated forms of dmrt2 in cos-7 cells ( figure 2b ) . full - length dmrt2 is localized primarily , but not exclusively , to vesicles outside the nucleus . a carboxy - terminal truncation containing the dna - binding domain is , however , concentrated almost entirely in the nucleus . these results are consistent with the idea that dmrt2 is cleaved from the membrane to produce a nuclear fragment . interestingly , transformer protein tra-2a , an indirect activator of mab-3 , has been identified recently as a membrane - tethered nuclear protein . thus , rip maybe a conserved mechanism common to sex determination in humans and worms . we used pfam   hidden markov models for 53 dna - binding domains ( see materials and methods ) to search all proteins in swiss - prot / trembl   and swisspfam protein databases . the 9,261 proteins identified by our search are presumed members of dna - binding protein families , and most are expected to be transcription factors . these proteins were then scored for the presence of one or more transmembrane segments using prediction programs phdhtm [ l8 ] , tmhmm , hmmtop   and psortii . only those proteins containing membrane - spanning helices predicted by at least three of the four programs were deemed significant in our analysis . by these stringent criteria , 76 proteins from 20 organisms and our analysis predicted a surprisingly large and diverse set of membrane - tethered dna - binding proteins . seventeen of the 53 dna - binding domains chosen for this analysis were represented in the final set of tmtfs . of these , the most abundant is the zf - c4 ( zinc - finger type c4 ) nuclear hormone receptor dna - binding domain , found in 14 proteins in caenorhabditis elegans and avian erythroblastosis virus . tmtfs in arabidopsis were the most diverse , and were associated with eight different dna - binding domains . all but two proteins have dna - binding domains that could be separated from the rest of the protein by a single hypothetical cleavage event , if singly predicted transmembrane segments are discounted ( figure 1 ) . dna - binding domains were also frequently juxtaposed to bipartite nuclear localization signals , suggesting that transmembrane and dna - binding domains in tmtfs are modular . thus , the overall topology of these proteins is consistent with other known tmtfs . c. elegans has an impressive 25 predicted tmtfs , suggesting that rip may be particularly important in the regulation of transcriptional responses in the worm . we deliberately used a stringent method to increase the likelihood of identifying only bona fide tmtfs and , as expected , most experimentally known tmtfs were detected by our analysis , including cadc , lzip , toxr   and all swiss - prot / trembl orthologs of srebp - l and srebp-2 . also found were several well - characterized proteins whose predicted membrane insertion had not been recognized . for example , the human doublesex - related protein dmrt2 , drosophila b - h2 ( barh2 ) protein , c. elegans unc-86 , and mouse oasis protein are predicted tmtfs . the transmembrane helix of atf6 was predicted by only two programs : psort and hmmtop . the immunoglobulin dna - binding domain ( tig ) of spt23 is found in both cell - surface proteins as well as transcription factors and was therefore excluded from the set of dna - binding domains . these results indicate that reducing the stringency of our prediction method will expand the number of predicted tmtfs . in some cases we found data in the literature to support our computational predictions . for example , cell - fractionation studies using antibodies directed at the carboxyl terminus of the chaperonin mtj1 showed that the full - length ( 62 kda ) protein exists in microsomes , whereas a smaller 42 kda form of the protein is found in the nucleus . the 42 kda species was hypothesized to represent a product of internal translation . because mtj1 contains putative myb dna - binding domains within the carboxy - terminal half of the protein , we re - examined the subcellular localization of carboxy - terminal - tagged mtj1 in cos-7 cells ( figure 2a ) . our results show clearly that full - length mtj1 is normally associated with the endoplasmic reticulum . in contrast , a truncated form mtj1 ( approximately 40 kda in size ) lacking the transmembrane segment accumulates in the nucleus . therefore , we propose that the 42 kda nuclear form of mtj1 observed in cells arises by cleavage of mtj1 from the membrane , rather than from aberrant translation of the mrna . dmrt2 , a human homolog of c. elegans mab-3 , was identified in our analysis as having a carboxy - terminal transmembrane segment ( figure 1 ) . dmrt2 has gained recent attention as a candidate gene for sex - reversal phenotypes in humans . to verify our prediction that dmrt2 is a membrane - tethered transcription factor , we examined the subcellular localization of full - length and truncated forms of dmrt2 in cos-7 cells ( figure 2b ) . full - length dmrt2 is localized primarily , but not exclusively , to vesicles outside the nucleus . a carboxy - terminal truncation containing the dna - binding domain is , however , concentrated almost entirely in the nucleus . these results are consistent with the idea that dmrt2 is cleaved from the membrane to produce a nuclear fragment . interestingly , transformer protein tra-2a , an indirect activator of mab-3 , has been identified recently as a membrane - tethered nuclear protein . thus , rip maybe a conserved mechanism common to sex determination in humans and worms . we have used computational methods to investigate the prevalence of membrane - tethered transcription factors . the identification of 76 predicted tmtfs by our method , and the supporting cell biology , indicate that membrane - tethering may be a common mechanism for regulating transcriptional responses . as stringent criteria were used to identify transmembrane segments and dna - binding domains , we believe that the actual number of tmtfs is likely to be much larger . compared to other signal transduction mechanisms , tethering transcription factors in the membrane provides an expeditious route to the nucleus in response to stimuli that must be communicated across a membrane . our understanding of this process will be enhanced as more tmtfs are studied and the signals for membrane cleavage and their proteases are discovered . pfam   hidden markov models for 53 dna - binding domains ( see dna - binding domains below ) were used to search proteins in swiss - prot / trembl ( october 2000 release ; 388,909 proteins ) with p - value < 0.0019 ( 0.01/53 ) . swisspfam proteins identified as having any of the 53 domains were also included in our analysis . default parameters were used for hmmtop , phdhtm [ l8 ] , and tmhmm ( version 2 ) . a higher stringency ( -5.0 ) than default was used for psort ii ( alom2 ) . transmembrane segments predicted by individual programs were considered overlapping if ten or more amino acids were shared by each segment . proteins containing transmembrane helices predicted by at least three of the four programs were included in the final set . three predicted tmtfs were discounted as false - positives on the basis of partial or complete overlap of transmembrane helices with other pfam domains ( o01612 , o23045 and q13771 ) . the following pfam models for dna - binding domains were used ( abbreviated as in pfam ) : 7 kda dna - binding ; ap2-domain ; arid ; asnc trans reg ; at hook ; arg represser ; b3 ; bah ; bro ; bac dna - binding ; basic ; bzip ; cbfb nfya ; csd ; cut ; copper - fist ; dm - domain ; e2f tdp ; fork head ; gata ; halz ; hlh ; homeobox ; hsf dna - binding ; hth 3 ; hth 4 ; hth 5 ; irf ; lexa dna - binding ; mbd ; metj ; myb dna - binding ; muts n ; myc - lz ; phd ; rfx dna - binding ; rhd ; runt ; sap ; sigma70 ; srf - tf ; stat ; sigma54 factors ; sigma70 ecf ; t - box ; tbp ; yeast dna - binding ; trans reg c ; zf ( zinc finger)-c2h2 ; zf - c2hc ; zf - c4 ; zf - nf - x1 ; zn - clus . full - length dmrt2 and mtj1 were generated by pcr using pfu polymerase ( stratagene ) and cloned directionally into bamhi / xbai sites of pcdna3 ( invitrogen ) . truncated mtj1 , in which an atg ( methionine ) was added immediately before amino acid 171 ( q61712 ) , was amplified from expressed sequence tag ( est ) ai790297 ( incyte genomics ) and a myc tag was added at the carboxyl terminus . full - length mtj1 was prepared by digesting clone ai790297 with xhoi , blunting ends , then digesting with ecori . this fragment was then cloned into pcdna3-mtj1 , which was digested with bamhi , blunt - ended , and digested with ecori . full - length and truncated dmrt2 ( at amino acid 180 ; q9y5r5 ) were amplified from est ai985131 ( incyte ) , and a myc tag was added at the amino terminus . standard deae transfections   of plasmids were done in cos-7 cells ( atcc ) and grown in 10% fbs / dmem . cells were fixed 72 h post - transfection in 3% pfa in pbs and myc tags were detected with mouse anti - myc antibodies ( neomarkers , fremont , ca ) and texas - red - x goat anti - mouse antibodies ( molecular probes , eugene , or ) using standard procedures . pfam   hidden markov models for 53 dna - binding domains ( see dna - binding domains below ) were used to search proteins in swiss - prot / trembl ( october 2000 release ; 388,909 proteins ) with p - value < 0.0019 ( 0.01/53 ) . swisspfam proteins identified as having any of the 53 domains were also included in our analysis . default parameters were used for hmmtop , phdhtm [ l8 ] , and tmhmm ( version 2 ) . a higher stringency ( -5.0 ) than default was used for psort ii ( alom2 ) . transmembrane segments predicted by individual programs were considered overlapping if ten or more amino acids were shared by each segment . proteins containing transmembrane helices predicted by at least three of the four programs were included in the final set . three predicted tmtfs were discounted as false - positives on the basis of partial or complete overlap of transmembrane helices with other pfam domains ( o01612 , o23045 and q13771 ) . the following pfam models for dna - binding domains were used ( abbreviated as in pfam ) : 7 kda dna - binding ; ap2-domain ; arid ; asnc trans reg ; at hook ; arg represser ; b3 ; bah ; bro ; bac dna - binding ; basic ; bzip ; cbfb nfya ; csd ; cut ; copper - fist ; dm - domain ; e2f tdp ; fork head ; gata ; halz ; hlh ; homeobox ; hsf dna - binding ; hth 3 ; hth 4 ; hth 5 ; irf ; lexa dna - binding ; mbd ; metj ; myb dna - binding ; muts n ; myc - lz ; phd ; rfx dna - binding ; rhd ; runt ; sap ; sigma70 ; srf - tf ; stat ; sigma54 factors ; sigma70 ecf ; t - box ; tbp ; yeast dna - binding ; trans reg c ; zf ( zinc finger)-c2h2 ; zf - c2hc ; zf - c4 ; zf - nf - x1 ; zn - clus . full - length dmrt2 and mtj1 were generated by pcr using pfu polymerase ( stratagene ) and cloned directionally into bamhi / xbai sites of pcdna3 ( invitrogen ) . truncated mtj1 , in which an atg ( methionine ) was added immediately before amino acid 171 ( q61712 ) , was amplified from expressed sequence tag ( est ) ai790297 ( incyte genomics ) and a myc tag was added at the carboxyl terminus . full - length mtj1 was prepared by digesting clone ai790297 with xhoi , blunting ends , then digesting with ecori . this fragment was then cloned into pcdna3-mtj1 , which was digested with bamhi , blunt - ended , and digested with ecori . full - length and truncated dmrt2 ( at amino acid 180 ; q9y5r5 ) were amplified from est ai985131 ( incyte ) , and a myc tag was added at the amino terminus . standard deae transfections   of plasmids were done in cos-7 cells ( atcc ) and grown in 10% fbs / dmem . cells were fixed 72 h post - transfection in 3% pfa in pbs and myc tags were detected with mouse anti - myc antibodies ( neomarkers , fremont , ca ) and texas - red - x goat anti - mouse antibodies ( molecular probes , eugene , or ) using standard procedures . we thank j. rine and o. kelly for critical comments on the manuscript , and d. he for assembling overlapping domains . pfam - predicted dna - binding domains , transmembrane segments and bipartite nuclear localization signals are shown for linear protein models and identified by swiss - prot / trembl accession number . colored icons represent various dna - binding domains . predicted transmembrane segments for each program are represented by a filled box . arrows in mtj1 and dmrt2 indicate sites for truncated protein localization experiments shown in figure 2 . orthologs of predicted tmtfs not shown are : luman ( q9ue77 homo sapiens ) , srebp-1 ( q60416 cricetulus griseus , q9wtn3 mus musculus , p56720 rattus norvegicus , q9xx00 caenorhabditis elegans ) , srebp-2 ( q9uh04 h. sapiens , q60429 c. griseus ) , and aflr reg ( p43651 aspergillus parasiticus ) . open reading frames ( orfs ) for o65420 , o43989 , q17928 were extended using additional nucleotide sequence available in the ncbi database ( indicated by stippled rectangles ) . subcellular localizations of predicted and truncated tmtfs in cos-7 cells were detected using anti - myc antibodies . full - length proteins are localized to intracellular membrane compartments , but truncated forms (  ) lacking predicted transmembrane segments accumulate in the nucleus .
backgroundsequestration of transcription factors in the membrane is emerging as an important mechanism for the regulation of gene expression . 
 a handful of membrane - spanning transcription factors has been previously identified whose access to the nucleus is regulated by proteolytic cleavage from the membrane . to investigate the existence of other transmembrane transcription factors 
 , we analyzed computationally all proteins in swiss - prot / trembl for the combined presence of a dna - binding domain and a transmembrane segment.resultsusing pfam hidden markov models and four transmembrane - prediction programs , we identified with high confidence 76 membrane - spanning transcription factors in swiss - prot / trembl . 
 analysis of the distribution of two proteins predicted by our method , mtj1 and dmrt2 , confirmed their localization to intracellular membrane compartments . 
 furthermore , elimination of the predicted transmembrane segment led to nuclear localization for each of these proteins.conclusionsour analysis uncovered a wealth of predicted membrane - spanning transcription factors that are structurally and taxonomically diverse , 56 of which lack experimental annotation . 
 seventy - five of the proteins are modular in structure , suggesting that a single proteolysis may be sufficient to liberate a dna - binding domain from the membrane . 
 this study provides grounds for investigations into the stimuli and mechanisms that release this intriguing class of transcription factors from membranes .