Báo cáo khoa học: A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein potx

A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein ´ ´ ´ Froilan Granero*, Fernando Revert, Francisco Revert-Ros, Sergio Lainez, Pilar Martınez-Martınez and Juan Saus ´ ´ Centro de Investigacion Prıncipe Felipe, Valencia, Spain Keywords Goodpasture antigen-binding protein; tumor necrosis factor; Goodpasture disease; bidirectional promoter; DNA polymerase j Correspondence ´ ´ J Saus, Centro de Investigacion Prıncipe Felipe, Avenida de la Autopista del Saler 16 C ⁄ En Proyecto, (Camino de las Moreras), 46013 Valencia, Spain Fax: +34 96 3289701 Tel: +34 96 3289680 E-mail: jsaus@ochoa.fib.es Present address *Center for Matrix Biology Vanderbilt University Medical Center, Nashville, TN, USA (Received July 2005, accepted 19 August 2005) doi:10.1111/j.1742-4658.2005.04925.x The Goodpasture antigen-binding protein, GPBP, is a serine ⁄ threonine kinase whose relative expression increases in autoimmune processes Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in autoimmune pathogenesis Here we show that COL4A3BP, the gene encoding GPBP, maps head-to-head with POLK, the gene encoding for DNA polymerase kappa (pol j), and shares with it a 140-bp promoter containing a Sp1 site, a TATA-like element, and a nuclear factor kappa B (NFjB)-like site These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFjB that is more efficient in the POLK direction Tumour necrosis factor cell induction is associated with Sp1 release, NFjB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction This is accomplished by competitive binding of Sp1 and NFjB to a DNA element encompassing a NFjB-like site that is pivotal for the 140-bp promoter to function Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA-like element but is devoid of the NFjB-like site, does not show transcriptional activity in transient gene expression assays Our findings identify a human-specific TNF-responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity Goodpasture disease and other more common natural autoimmune disorders have been described only in humans Goodpasture antigen-binding protein (GPBP) is a serine ⁄ threonine kinase that specifically targets a motif in the NC1 domain of the human a3 chain of type IV collagen (the Goodpasture autoantigen), which is highly divergent and does not exist in other species or human homologous domains [1,2] GPBP shows preferential expression in cells and in association with tissue structures that undergo common autoimmune attacks – including the glomerular basement membrane targeted in Goodpasture disease [2] Moreover, an augmented expression of GPBP with respect to its alternative spliced variant GPBPD26 has been associated with a number of autoimmune responses [2], all which suggests that GPBP is relevant in human autoimmune pathogenesis COL4A3BP, the gene encoding GPBP, is headto-head with POLK, the gene encoding DNA polymerase kappa (pol j; GenBank accession number AB036934) Pol j and its prokaryote counterpart, pol IV, display low fidelity, moderate processivity, and Abbreviations EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPBP and GPBPD26, Goodpasture antigen-binding protein and its alternative splicing variant devoid of the exon-encoded 26-residue motif; NFjB, nuclear factor kappa B; NR, nonrelevant; pol j, DNA polymerase kappa; siRNA, small interfering RNA; TAF, TBP-associated factor; TAF, TBP-associated factors; TBP, TATA-binding protein; TNF, tumor necrosis factor FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS 5291 TNF transcriptional induction of GPBP extend mispaired DNA by misaligning primer–template to generate )1 or )2 frameshift products [3–7] Furthermore, pol j can bypass DNA lesions in both error-prone and error-free manners [7–10] All of this suggests that pol j is a DNA polymerase that not only has a role in the cellular response to DNA damage, but also plays a part in spontaneous mutagenesis by facilitating base pairing at aberrant replication forks Tumour necrosis factor (TNF)-a and -b are two different cytokines with similar biological effects that are primarily secreted by macrophages and Th1 cells in response to various inflammatory stimuli (reviewed in [11]) Of all the known biological effects of TNF, the cytotoxicity seems to be the most prominent, and thus TNF is the mediator whereby cytolytic immune cells induce fatal injury to their targets However, overproduction of TNF in concert with other cytokines often has negative consequences for the host, for example in various autoimmune and immune-mediated rheumatologic disorders Here we show that TNF increases the mRNA levels of GPBP and induces COL4A3BP expression from a promoter that in its constitutive mode transcribes POLK more efficiently This is accomplished, at least in part, by Sp1 release from – and NFjB recruitment to ) a bidirectional complex that binds to several DNA elements including one novel element that encompasses a nuclear factor kappa B (NFjB)-like site and supports competitive binding of the two transcription factors Moreover, we show that the mouse DNA counterpart is devoid of this versatile element, thereby uncovering major differences in GPBP expression between human and mouse that may be important in understanding human-specific autoimmune pathogenesis Results Tumour necrosis factor increases the mRNA levels of GPBP in human cells Human cultured HEK293 and hTERT-RPE1 cells were induced with human recombinant TNF-a, and the levels of GPBP mRNA were estimated by a reverse transcriptase-real time PCR approach (DDCt) (Fig 1) We observed an overall increase in the mRNA levels of GPBP in response to TNF in both cell types However, the mRNA levels increased more promptly in hTERT-RPE1 reaching three- to fourfold after h of induction whereas h of induction were required to reach 2- to 2.5-fold levels in HEK293 cells Our findings suggest that TNF induces GPBP transcription in human cells 5292 F Granero et al Fig TNF increases the mRNA levels of GPBP in cultured human cells HEK293 (293) or hTERT-RPE1 (RPE1) cells were cultured in the presence or in the absence of TNFa during the indicated periods of time, disrupted, and total mRNA extracted and subjected to reverse transcriptase-coupled real-time PCR to estimate the levels of mRNA by the DDCt method The values represent fold of induced vs noninduced cells after normalization with the corresponding values for GAPDH We represent the mean of three independent experiments carried out in duplicate ± SD The mRNA levels of GAPDH were not affected by cytokine induction Identification of a 140-bp bidirectional promoter in the intergene region of COL4A3BP and POLK Using cDNA mapping we identified a 140-bp fragment in the intergene region of COL4A3BP and POLK (Fig 2A) To investigate the presence of a bidirectional promoter in this 140-bp DNA region, we cloned each of the two orientations of a 772-bp fragment encompassing the region of interest in pFGH (LpromGPBP and LpromPolj) and assessed its capacity to drive heterologous gene expression in mammalian cells (Fig 2B) The 772-bp fragment efficiently promoted transcription in each direction, 21-fold for COL4A3BP and 25-fold for POLK When we assessed the transcriptional activity of the 140-bp region (SpromGPBP and SpromPolj), we observed a reduction in the activity that was more evident for COL4A3BP than for the POLK direction, a 45% reduction vs 18% This indicates that although the 140-bp region contains the core of a bidirectional transcriptional unit and the structural requirements for divergent transcription, there FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al TNF transcriptional induction of GPBP A B C Fig Identification of a bidirectional promoter for human POLK and COL4A3BP and transcription start sites mapping (A) The one-strand sequence between ON-GPBP-18m and ON-GPBP-5c is written in upper case Indicated are the position and sequence of oligonucleotides used for DNA isolation and the restriction sites used to generate constructs for experimental procedures in (B) and (C) Open and closed boxes contain DNA sequences present in the indicated pol j (GenBank accession number AF163570) and GPBP (GenBank accession number AF136450) cDNA clones of which the 5¢-ends and transcription direction are denoted by arrows Highlighted in grey is the 140-bp fragment present in Sprom constructs Bent arrows indicate the transcription start sites mapped in (C) (B) Cells were transfected with either Lprom (L) or Sprom (S) constructs along with a b-galactosidase-expressing vector Results are expressed as the quotient (fold) of the reporter gene expression of the promoter constructs vs empty vector previously normalized with the corresponding b-galactosidase expression values Values represent the mean of two independent experiments carried out in duplicate ± SD (C) Antisense ribonucleotide probes (2 · 105 cpm) representing the indicated DNA regions were mock digested (1) or digested with RNase in the presence of yeast (2) or human (3) total RNA extracted from hTERT-RPE1 cells (COL4A3BP) or from HEK293 (POLK) The amounts of RNA used were 40 lg (COL4A3BP) or 80 lg (POLK) After digestion of the single-strand RNA the mixtures were analysed by denaturing PAGE and autoradiographed The numbers and bars indicate the size in nucleotides and position of radiolabelled ribonucleotide markers FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS 5293 TNF transcriptional induction of GPBP are regulatory elements in the flanking regions that modulate both transcriptional activity and direction F Granero et al A Mapping of transcriptional start sites for COL4A3BP and POLK We used RNA-protection assays to map the transcriptional start sites for each gene (Fig 2C) When 32 P-labelled RNA probes representing the antisense strand of COL4A3BP or POLK between the ApaI and EclXI sites (Fig 2A) were hybridized with human RNA, three and two fragments, respectively, were protected from RNase digestion (Fig 2C), thereby revealing the existence of multiple mRNA species for GPBP and pol j To further map the 5¢ end of the individual mRNAs, we similarly assayed antisense RNA probes representing DNA sequences which extend into the corresponding gene (BamHI ⁄ ON-GPBP-5c and ONpolk-2m ⁄ EclXI) These resulted in larger protected fragments displaying sizes that were consistent with the extended sequences (Fig 2C) and thus mapping the 5¢ end of the different mRNA species at the positions indicated in Fig 2A All of these findings suggest that the DNA region identified by genomic cDNA mapping is at the intergene region and contains the structural requirements for bidirectional transcription B C Identification and characterization of transcriptional DNA elements in the 140-bp promoter Using matinspector V2.2 from the transfac 4.0 program [12], we have identified three transcriptional elements in the DNA region of interest Two elements commonly found associated with transcriptional initiation in TATA-less promoters, a canonical Sp1 binding site and a TATA-like element [13–15], and a NFjB-like binding site for transcriptional regulation (Fig 3A) The contribution of individual DNA elements in transcription was first assessed in 140-bp promoter constructs in which elements expected to be associated with transcriptional initiation were deleted (Fig 3B) Deletion of each individual element had negative consequences on transcription although impacted differently each orientation Deletion of the Sp1 site (DSp1) resulted in major transcription impairment in both directions ( 60–70%) whereas TATA-like element deletion (DTATA) significantly reduced transcription in the COL4A3BP direction ( 60%) but had a lesser negative effect on the POLK direction ( 30%) Finally, double deletion (DSp1 ⁄ TATA) reported additional transcriptional activity reduction only in the COL4A3BP direction, in which case the promoter dis5294 Fig Identification of transcriptional DNA elements in 140-bp promoter (A) The one-single strand sequence of the 140-bp promoter is written in upper case and underlined are the DNA elements identified using the MatInspector V2.2 from the TRANSFAC 4.0 program (B,C) Cells were transfected with SpromGPBP or SpromPolj (WT) or with mutants thereof in which the NFjB-like (NFjB) and ⁄ or Sp1 (Sp1) binding sites and ⁄ or TATA-like element (TATA) were deleted (D) Transcriptional activities were normalized as in Fig 2B and results are expressed as percent activity with respect to WT sequence, which was set at 100%, and are the mean ± SD of three experiments carried out in duplicate played a transcriptional activity ( 20%), which was only slightly above the values observed for the empty vector (7–12%) This suggests that although the two elements are used in the two directions, COL4A3BP transcription depends more on TATA-like element than POLK, which depends mainly on the Sp1 site The evidence also suggests that the TATA-like element and the Sp1 FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al site are coordinated when transcribing COL4A3BP as the relative contribution of each element to total activity is very similar and too high to be fully independent To assess the role of the NFjB-like site in transcription, the multiple promoter constructs were further deleted and similarly assayed (Fig 3C) Deletion of the NFjB-like site (DNFjB) greatly reduced transcription in the COL4A3BP direction (80%) and little in the POLK direction ( 30%) Additional deletion of the TATA-like element (DNFjB ⁄ TATA) did not reduce transcriptional activity, while additional deletion of the Sp1 site (DNFjB ⁄ Sp1) rendered the promoter virtually inactive in the COL4A3BP direction and with an activity in the POLK direction slightly above empty vector Triple deletion mutants were not significantly different from DNFjB ⁄ Sp1 mutants Collectively these results suggest that NFjB- and TATA-like sites are coordinated elements that account for  80% of transcription in the COL4A3BP direction whereas the Sp1 site accounts for the remaining  20% In contrast the Sp1 site accounts for  70% of transcriptional activity in the POLK direction and the remaining  30% depends on NFjB- and TATA-like sites Of special interest was the observation that the double deletion DNFjB ⁄ TATA rendered a promoter with transcriptional activities in the POLK direction that were similar and even higher than the wild-type promoter, while single deletions mutants (DNFjB or DTATA) rendered promoters with less activity ( 30% reduction), suggesting that Sp1 site and coordinated NFjB ⁄ TATA-like elements compete for transcription factors Sp1- and NFjB-based transcriptional complexes coexist and compete for DNA elements in the 140-bp promoter Collectively our data suggest that there are two bidirectional transcriptional complexes, one binding to the Sp1 site which is more efficient in the POLK direction and another operating through NFjB-like and TATA-like sites (NFjB-TATA-dependent) comparatively more efficient in the COL4A3BP direction Both, however, coexist and compete for the promoter suggesting that the two complexes bind to all three sites To test this hypothesis we performed electrophoretic mobility shift assay (EMSA) using nuclear extracts and double-stranded synthetic oligonucleotides representing the DNA elements and flanking regions of interest (NFjB-L, SP1 and TATA-L) (Fig 4) Individual 32P-labelled DNAs were bound to nuclear proteins FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS TNF transcriptional induction of GPBP Fig Mapping transcription complex binding sites in the 140-bp promoter using EMSA On the left in the composite and in upper case is the two-strand nucleotide sequence of the 32P-labelled probe used in EMSA as shown on the right Each sequence represents the indicated element (parallel lines) and flanking regions in the 140-bp promoter (NFjB-L, SP1, and TATA-L) The 32P-labelled DNA fragments were incubated in the presence (+) or in the absence (–) of nuclear extracts (NE) and the indicated competitors (Comp) NR is a nonrelevant DNA The resulting complexes were analysed by nondenaturing PAGE and autoradiographed With arrows we indicate the position of the retarded autoradiographic bands referred to in the text In the composite, the individual samples from the same study and exposure have been placed in a different order than in the original gel for illustrative purposes to generate complexes whose assembly was efficiently inhibited by the corresponding unlabelled DNA but not by similar concentrations of a nonrelevant DNA (NR), suggesting that nuclear proteins form complexes with DNA in a sequence-dependent manner Interestingly, individual unlabelled DNA elements showed capacity to inhibit or alter the binding of nuclear proteins to the other 32P-labelled elements In this regard, NFjB-L was the most efficient as it completely inhibited the formation of one [32P]SP1 ⁄ protein complex (top arrow), strongly inhibited another (bottom 5295 TNF transcriptional induction of GPBP arrow), and fully inhibited two of the three complexes associated with [32P]TATA-L (top and bottom arrows) In contrast, SP1 showed the least effect on protein binding to other elements and inhibited the formation of one of the complexes associated to [32P]NFjB-L (top arrow) without significantly impacting the other (bottom arrow), and only caused an alteration in the relative abundance of individual complexes associated with [32P]TATA-L (top and bottom arrows) Finally, TATA-L efficiently inhibited formation of one complex associated with [32P]NFjB-L (bottom arrow) without significantly impacting the other (top arrow) and partially inhibited formation of the two [32P]SP1 complexes (top and bottom arrows) Of special interest was to find that [32P]NFjB-L associates with nuclear proteins to form two types of independent complexes, one that can (bottom) and another that cannot (top) be assembled in the presence F Granero et al of SP1, suggesting that Sp1 and NFjB transcription factors compete for NFjB-L The evidence also suggests that there are two complexes that bind to all three DNA elements, although in different manners Sp1-based complex preferentially binds to SP1 and displays lower affinity for NFjB-L and still much lower for TATA-L The NFjB-based complex preferentially binds to NFjB-L and displays lower but similar affinity for SP1 and for TATA-L Tumour necrosis factor induces asymmetric transcription from the 140-bp promoter To assess TNF induction of the 140-bp promoter, we performed transient gene expression assays (Fig 5A) Tumour necrosis factor cell stimulation resulted in promoter induction, but this was more evident in the COL4A3 BP (three- to fourfold) than in the POLK A B Fig TNF induces transcription from the 140-bp promoter (A) Cells were transfected with SpromGPBP or SpromPolj along with a b-galactosidase-expressing vector and were induced or not with TNF Results are expressed as the quotient (fold) of the reporter gene expression of the induced vs noninduced cells after value normalization with the corresponding b-galactosidase expression values We represent the mean of four independent experiments carried out in duplicate ± SD (B) Cells were transfected with SpromGPBP or SpromPolj (WT) or with mutants thereof, in which the NFjB-like (NFjB) and ⁄ or Sp1 (Sp1) binding sites and ⁄ or TATA-like element (TATA) were deleted (D), were induced with TNF (+) or not induced (–), and transcriptional activities estimated and expressed as in Fig We represent the mean of three experiments carried out in duplicate ± SD 5296 FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al (one- to twofold) direction Similar results were obtained when promoter transactivation was assessed in NIH3T3 (shown) or in HEK293 cells using a different reporter gene expression system (data not shown) To determine the contribution of individual DNA elements in transcriptional induction, we performed similar assays on induced and noninduced cells using specific deletion mutants (Fig 5B) We found no major differences in the overall relative contribution of each of the two main transcriptional activities (NFjB dependent and Sp1 dependent) between constitutive and induced modes of expression in the POLK direction In contrast, deletion of NFjB-like (DNFjB) or Sp1 (DSp1) sites had significantly less negative impact on induced than on constitutive expression in the COL4A3BP direction, although double deletion (DNFjB ⁄ Sp1) was still very efficient at reducing transcription to values slightly above background Additional deletion of the TATA-like element (DNFjB ⁄ Sp1 ⁄ TATA) did not further reduce transcriptional activity in the COL4A3BP direction, but double and triple mutants had an unusual induction in the POLK direction that was not observed in single mutants These results suggest that TNF induces transcription from the 140-bp promoter preferentially in the COL4A3BP direction involving the same DNA elements than when transcribing in a constitutive mode TNF induction of the 140-bp promoter involves Sp1 release and NFjB recruitment from a transcriptional complex that binds to all three elements To further characterize the molecular mechanism of TNF induction, the transcriptional complex associated with the 140-bp promoter in each expression mode was isolated and analysed by western blot using specific antibodies (Fig 6A) The transcriptional complex representing constitutive expression contained abundant Sp1 and traces of NFjB which were only detectable after longer developing times (not shown), whereas the transcriptional complex representing induced expression contained relatively less Sp1 and more NFjB (140-bp), thereby suggesting that TNF induction is mediated by Sp1 release from – and NFjB recruitment to ) the transcriptional complex We obtained similar conclusions when transcription complexes were purified from nuclear extracts of NIH3T3 cells (data not shown) Major transcriptional induction occurs in the COL4A3BP direction which depends mainly on a NFjB-like site To explore the mechanism of induction FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS TNF transcriptional induction of GPBP A B Fig Characterization of transcription complex associated to the 140-bp promoter isolated by affinity chromatography procedures (A) At the top we represent the 140-, 76- or 26-bp promoters with horizontal bars and recognized transcriptional elements therein in black At the bottom, nuclear extracts from noninduced (–) or TNFinduced (+) cells were subjected to affinity chromatography on columns containing the indicated promoter fragments and the bound proteins eluted and analysed by western blot using antibodies specific for the indicated transcription factors (B) The indicated amounts of nuclear extracts (NE) from noninduced (–) or TNFinduced (+) cells or proteins thereof bound to the indicated DNA fragments were analysed by western blot using antibodies specific for the indicated transcription factors further, we purified and similarly analysed transcription complexes bound to different promoter fragments and to the corresponding fragments devoid of the NFjB-like site (Fig 6A) Deletion of the NFjB-like site yields a promoter that binds transcription factors in a similar fashion to the 140-bp promoter (140bpDNFjB) A 76-bp promoter devoid of the TATA-like element (76-bp) displayed a relatively higher binding capacity for Sp1 than the 140-bp promoter, but induc5297 TNF transcriptional induction of GPBP tion was still associated with a net Sp1 release and NFjB recruitment When the NFjB-like element was deleted in the 76-bp promoter (76-bpDNFjB) the Sp1 binding pattern remained essentially unchanged but NFjB recruitment associated with induction was significantly impaired In contrast, a promoter only containing the NFjB-like site (26-bp) showed a reduced Sp1 binding capacity, and the recruitment of NFjB upon induction was not associated with Sp1 release Finally, specific deletion of the NFjB-like binding site from the 26-bp promoter resulted in a DNA fragment without capacity to bind either Sp1 or NFjB (26bpDNFjB) Subsequently, the contribution of individual DNA elements in transcriptional induction was also investigated (Fig 6B) We first determined the consequences of induction on the nuclear levels of specific transcription factors and found that TNF increased the nuclear levels of all members of the NFjB family and slightly decreased the presence of Sp1 in the nucleus, while the levels of TATA-binding protein (TBP), a general transcription factor critical for transcription preinitiation complex assembly, remained virtually unchanged Transcriptional complexes associated with individual DNA elements from noninduced nuclear extracts contained abundant Sp1 and only traces of individual NFjB members Furthermore, the transcriptional complex associated with NFjB-L contained traces of TBP whereas SP1 and TATA-L transcriptional complexes contained abundant TBP Cell induction resulted in recruitment of virtually all NFjB transcription factor members into each individual transcriptional complex without evident reduction in the amount of Sp1 and TBP The minor differences in the content of NFjB members among individual transcription complexes probably reflect different affinities of a common transcriptional complex for individual elements along with different protein–protein interactions, rather than different transcription complexes being assembled in the 140-bp promoter Collectively our data reveal the existence of at least two functionally different transcriptional complexes that bind to all three DNA elements, one containing Sp1 and little NFjB which represents the constitutive mode of expression, and the other containing abundant NFjB and comparatively less Sp1 which represents the induced mode Our findings also suggest that the Sp1 binding site is most critical for Sp1 recruitment whereas the NFjB-like binding site is the critical element for NFjB recruitment Consequently, net loss of Sp1 and gain of NFjB during induction requires both NFjB-like and Sp1 sites Our data also provide further evidence for the NFjB-like site to be a regulatory ele5298 F Granero et al ment bearing binding sites for NFjB and Sp1, and for the Sp1 site and TATA-like element to contain transcriptional initiators NFjB-like binding site is critical for adequate positioning of Sp1 in both constitutive and induced transcription complexes Our studies also reveal the existence of two major binding sites for Sp1 located at NFjB-like and Sp1 sites (vide supra) To further explore the role of each site in Sp1 binding and assembly, we used Sp1 antibodies (aSp1) and deletion mutants of the 76-bp promoter in EMSA (Fig 7) Nuclear proteins Fig NFjB-like site is critical for adequate Sp1 assembly in the transcription complex 32P-labelled 76-bp promoter or mutants thereof in which the NFjB-like (NFjB) and ⁄ or Sp1 (Sp1) binding sites were deleted (D), were incubated in the presence (+) or in the absence (–) of nuclear extracts (NE) from TNF-induced (+) or noninduced (–) cells and Sp1 specific antibodies (aSp1) The resulting complexes were analysed by nondenaturing PAGE and autoradiographed for 16 h (top row) or for 40 h (bottom row) We denote by arrows the supershifted or inhibited autoradiographic band associated with antibody presence FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al from either induced or noninduced cells were bound to 32P-labelled 76-bp promoter retarding its migration in PAGE (gel shift) in a similar fashion The presence of aSp1 in the binding mixtures resulted in a gel supershift that was more evident in noninduced nuclear mixtures (arrow in 76-bp) Deletion of NFjB-like binding site (76-bpDNFjB) resulted in a promoter that still bound nuclear proteins; however, aSp1 inhibited protein–DNA complex formation (gel shift inhibition), rather than inducing a gel supershift (arrow), suggesting that transcription factor assembly is largely defective In contrast, deletion of the Sp1 site yielded a promoter (76-bpDSp1) that bound nuclear proteins and aSp1 in a similar fashion as the 76-bp promoter (arrow) Although in this case, the promoter appears to bind Sp1 less efficiently (longer exposure times were required to visualize antibodydependent supershift bands) and the relative abundance of supershift bands was similar between noninduced and induced nuclear extracts Finally, double deletion resulted in a DNA that still formed complexes with nuclear proteins, but these were not reactive with the specific antibodies (76bpDNFjB ⁄ DSp1) These results suggest that in the transcription of the 140-bp promoter the Sp1 site is critical for effective Sp1 recruitment and release whereas the NFjB-like site is critical for adequate positioning of Sp1 in the transcriptional complex Sp1 and NFjB are components of the transcriptional complex associated with the 140-bp promoter ex vivo To investigate the physiological relevance of our findings mRNA silencing (expression of siRNA), western blot and ChIP assays were combined to investigate ex vivo binding of Sp1 and NFjB into the 140-bp promoter (Fig 8) Relevant antibodies (aSp1, ap65) specifically precipitated the 140-bp promoter from noninduced or induced cells as PCR procedures efficiently amplified the DNA region of interest from the corresponding immunoprecipitates but not from precipitates obtained with irrelevant antibodies (aNR) However, aSp1 was relatively more efficient in precipitating the 140-bp promoter from the chromatin of noninduced cells whereas ap65 was relatively more efficient at precipitating it from the chromatin of induced cells As expected, expression of individual siRNAs resulted in substantial reduction in the cellular levels of the corresponding transcription factors (Immunoblot); and consequently, in the capacity of the corresponding FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS TNF transcriptional induction of GPBP Fig Ex vivo binding of Sp1 and NFjB to the 140-bp promoter HEK293 cells were subjected to transfection in the absence (–) or in the presence of the indicated small interfering RNA (siRNA) and either TNF-induced (+) or not induced (–) and used for either western blot (Immunoblot) or ChIP assays using the indicated specific antibodies (aSp1 or ap65) Similar amounts of individual immunoprecipitates were subjected to PCR and resulting mixtures were analysed by agarose gel electrophoresis and ethidium bromide staining (ChlP) For control purposes, a nonrelevant antibody was included in the ChIP assays and the corresponding immunoprecipitates (aNR) and supernatants (DNA input) PCR-amplified and analysed in parallel specific antibodies to precipitate the 140-bp promoter (ChIP) This suggests that reduction of cellular levels of individual transcription factors efficiently reduces transcription factor–promoter binding Interestingly, reduction in the expression of one transcription factor affected promoter precipitation by the other transcription factor-specific antibodies, although in a different manner depending on expression mode In noninduced cells, reduction of cellular levels of one transcription factor was associated with increased promoter precipitation by the other transcription factor-specific antibodies This effect was more evident when reducing the expression of Sp1 than when reducing the expression of p65 in consonance with the prevalence of Sp1 vs p65 in the constitutive mode of transcription In contrast, in TNF-induced cells, reduction in the level of expression of one individual transcription factor was associated with a major reduction in promoter precipitation by the other transcription factor-specific antibodies This effect was more evident when reducing the expression of p65 than when reducing the expression of Sp1 in consonance with the prevalence of p65 vs Sp1 in the induced mode of transcription These findings reveal that the two transcription factors are part of the transcriptional machinery associated with the 140-bp promoter ex vivo, although Sp1 and p65 are more prominent in constitutive and induced modes of expression, respectively 5299 TNF transcriptional induction of GPBP This supports the idea that induction associates with Sp1 release from – and NFjB recruitment to ) the transcriptional complex However, there are major differences between constitutive and induced transcriptional complexes other than the relative abundance of one or the other transcription factor Thus binding of Sp1 and NFjB appears to be independent and competitive in noninduced cells and mutually dependent and coordinated in induced cells A B Fig A DNA element encompassing NFjB-like binding site is critical for transcriptional activity and TNF responsiveness of the 140bp promoter (A) the alignment of nucleotides 582–900 of GenBank accession number AF315603 and nucleotides 4557244–4557527 of GenBank accession number NT_039590.1, respectively, containing the human and mouse POLK ⁄ COL4A3BP intergene regions are shown in upper case Boxed are the human 140-bp promoter and the corresponding aligned mouse sequence Highlighted are the nucleotides identical between mouse and human DNA and underlined are human DNA elements The head-to-head arrows are written over the 7-bp palindrome flanking the triplet-in-tandem sequence (GGAGGA) With a vertical bar and an arrow, we indicate one end of the 127-bp promoter and the direction towards the other end, respectively (B) Cells were transfected with the pFGHbased constructs containing the promoter in the indicated transcriptional orientation, and either induced with TNF or not Results are expressed as counts per minute after value normalization with the corresponding b-galactosidase activity Shown are the results of one experiment carried out in duplicate ± SD Similar results were obtained in three independent experiments 5300 F Granero et al A DNA element encompassing the NFjB-like site is missing in the mouse DNA counterpart that shows no transcriptional activity Alignment of mouse and human COL4A3BP ⁄ POLK intergene regions reveals high degree of identity and the presence of several gaps in mouse genome including one major gap of 27-bp encompassing the NFjB-like site and its flanking regions (Fig 9A) To assess whether sequence differences had consequences in transcriptional activity, the mouse DNA region representing the 140-bp human unit (105-bp) was cloned in the two orientations in pFGH and its capacity to drive heterologous gene expression was assessed (Fig 9B) The mouse 105-bp DNA fragment had no significant transcriptional activity in either direction or mode of expression in experimental conditions in which human 140-bp was fully active Taking a closer look at the human-exclusive sequence, we found that the NFjB-like site is the core of a 20-bp sequence consisting of a 7-bp palindrome centred by a tripletin-tandem repeat (head-to-head arrows in Fig 9A) containing a recognition site for BamHI endonuclease We took advantage of such a restriction site and generated pFGH-based constructs in which half of the 20-bp sequence was deleted (127-bp, Fig 9A) Interestingly, the 127-bp construct showed no activity in the COL4A3BP direction and showed only residual activity when transcribing in the POLK direction (Fig 9B) Collectively our data suggest that bidirectional transcription from the human 140-bp DNA region depends mostly on a DNA element containing binding sites for Sp1 and NFjB which is critical for coordinated Sp1 release and NFjB recruitment mediating TNF induction Discussion In eukaryotes, TBP and TAFs (TBP-associated factors) are general transcription factors that constitute TFIID, a multisubunit complex required for RNA polymerase II positioning at either TATA box-containing or TATA-less promoters [15,16] The evidence supports that the canonical TATA element (TATAAA) mediates TFIID recruitment through TBP binding In contrast, in TATA-less promoters TFIID recruitment occurs through TAF binding to DNA sequences encompassing a transcription start site (initiator) In the latter, effective transcription requires the intervention of other nongeneral transcription factors which, in turn, reflect the level of transcription and cell transcription factor repertoire [17–19] Many housekeeping FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al transcriptional units, including bidirectional, often are devoid of a canonical TATA element and are transcribed from multiple start sites [14,20,21], suggesting that regulatory nongeneral factors are determinant to define transcription start site usage in these genes We have mapped three major transcriptional start sites for COL4A3BP and two for POLK; however, cDNA cloning approaches identify multiple mRNA species initiating between these sites (GenBank accession numbers AF163570 for POLK and BI826689, BI831357, BM827753, BI911338, BI599774 for COL4A3BP) Interestingly, BI826689 and BI831357 initiate in Sp1 flanking regions and BM827753, BI911338 and BI599774 initiate in the TATA-like element and flanking regions Among the remaining cDNAs species identified (GenBank accession numbers BI860449, BQ899288, BQ707890, BG828192, BG827803, BF310812, BF306225, CN351006, BI760500, CA397804, BM171788, BI093981, BU855806, BE734230, BE019579, BE272872, BE732391, BE278882, AA158617, AA121104, BE278407, BQ068905, BE734230), all of which derive from COL4A3BP, site b appears to be the most commonly used for initiation These observations support that the 140-bp promoter represents one of various alternative ways in which the COL4A3BP ⁄ POLK intergene region can be used for divergent transcription and for regulating GPBP and pol j expression The latter is accomplished from the 140-bp promoter by competitive assembly of Sp1 and NFjB into a bidirectional transcriptional complex that binds to three DNA elements (NFjB-like, Sp1 and TATA-like) As the NFjB-like site renders the 140-bp region competent for transcription and is the only element supporting direct Sp1 and NFjB binding – we found that unlabelled validated consensus sequences for NFjB efficiently inhibited nuclear protein binding to [32P] NFjB-L, but not to [32P]SP1 or [32P]TATA-L in EMSA – it seems that bidirectional transcription from the 140-bp region depends mainly on competitive Sp1 and NFjB binding to the NFjB-like site It remains to be determined, however, whether the two transcriptional factors compete for assembly in a single transcription complex or they exist in separate complexes which compete for the promoter Tumour necrosis factor induces COL4A3 BP expression by increasing the nuclear levels of NFjB but also by promoting the structural linkage of the two transcription factors, suggesting that additional signals other than the relative nuclear abundance of Sp1 and NFjB are needed for regulating transcriptional orientation FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS TNF transcriptional induction of GPBP A number of natural autoimmune responses mediating low or high frequency disorders (e.g Goodpasture disease or multiple sclerosis) have been described only in humans, and counterparts in the animal world have not yet been reported This observation supported initial studies directed to identify human-specific biological features in the context of Goodpasture disease, which resulted in the identification of a unique phosphorylatable region in the human autoantigen [22,23] and subsequent discovery of GPBP, a nonconventional protein kinase that specifically targets the autoantigen unique region [1,2] Now, through the characterization of a DNA element without counterparts in mouse, chicken or rat genomes (GenBank accession numbers NW_060733 and NW_047617), we identified a TNF-responsive human-specific transcriptional unit and provided new insights on GPBP expression that may be of relevance to understanding human-specific autoimmune pathogenesis During maturation from basal to peripheral strata, keratinocytes undergo an apoptosis-dependent differentiation process that is controlled, at least in part, by Sp1 and NFjB-related factors [24–26] Keratinocytes from patients suffering skin autoimmune processes show an increased sensitivity to UV-induced apoptosis and a premature apoptosis at the basal keratinocytes [27–29] Goodpasture antigen-binding protein is highly expressed in peripheral keratinocytes in normal epidermis but is overexpressed in keratinocytes expanding from basal to peripheral strata in autoimmune processes [2] Finally, it has been reported that altered autoantigens, including phosphorylated versions thereof, are released from keratinocytes undergoing apoptosis [28,30] All of this suggests that in keratinocytes NFjB-induced GPBP expression is part of a desired cell death program that operates illegitimately during autoimmune pathogenesis, in which aberrant counterparts of critical cell components are generated The head-to-head arrangement of COL4A3BP and POLK through a bidirectional promoter suggests that the products encoded by these genes are partners in specific cell programs Consistently, we have found a coordinated augmented expression of COL4A3BP and POLK in skin undergoing an autoimmune attack, and we have cloned a previously unrecognized alternatively spliced isoform of 76-kDa, pol j76 (GenBank accession number AF315602), whose relative expression with respect to pol j was found to be increased in keratinocytes and in skin autoimmune processes (unpublished observations) Pol j76 may represent the version of pol j in a somatic mutation-based strategy 5301 TNF transcriptional induction of GPBP to generate aberrant counterparts of critical cell components as part of a common cell death program that is relevant in both keratinocyte maturation and autoimmune pathogenesis Experimental procedures Synthetic oligonucleotides ON-GPBP-6c, 5¢-CTCGCTCGCCCAGGGAAGGAAAA GGGAAAAGAAGGGA-3¢; ON-GPBP-18 m, 5¢-GGCAT GGTTAACGTGGTTCTC-3¢; ON-XbaG ⁄ Bpro1m, 5¢-GA CTCTAGAGGGTTCGGGAGGAGGATCCCG-3¢; ONXbaG ⁄ Bpro1c, 5¢-GACTCTAGACTGGCCCACTATTTA CCCTCC-3¢; ON-GPBP-5c, 5¢-CTCGATGCCAATTTCAA ATAGGGAA-3¢; ON-polj-2m, 5¢-GACAAGCCGCCCTG GAAAGCAGGCCC-3¢; ON-polj-3m, 5¢-GCAGCACAGC TGCATCCCTACCCCGCCCTCTC-3¢; ON-SP1Del, 5¢-CG CCGGGAGGGGGACGTAGTGGGGGAGAAT-3¢; ONNFjBmut, 5¢-TCTAGAGGGTTCGGGAGAAGGCTCGG CGTGTCG-3¢; ON-TATADel, 5¢-CAGGGGAGGGGAG GGGTGGGCCAGTCTAGA-3¢; ON-SP1-3m, 5¢-GGGGG ACGGGGCGGGGAGTAGTGG-3¢; ON-SP1-3c, 5¢-CCA CTACTCCCCGCCCCGTCCCCC-3¢; ON-TATA-1m, 5¢-GA GGGGAGGGTAAATAGTGGGCCAG-3¢; ON-TATA-1c, 5¢-GTGGCCCACTATTTACCCTCCCCTC-3¢; ONNFjBmut-2c, 5¢-GCCTTCTCCCGAACCC-3¢; ON-NFjBmut-2m, 5¢-GGGTTCGGGAGAAGGC-3¢; ON-NFjB-1m, 5¢-GGGTTCGGGAGGAGGATCCCGAAGGC-3¢; ONNFjB-1c, 5¢-GCCTTCGGGATCCTCCTCCCGAACCC-3¢; ON-SP1del-c, 5¢-CCCCCACTACGTCCCCCTCCC-3¢; ONPromMouse-F, 5¢-GACTCTAGAGGGAGCGTCGCGAG CCGCCGGGAG-3¢; ON-PromMouse-R, 5¢-GACTCTAG ACGGGTCCACTATTTACCCTCCCTC-3¢; ON-EcoRIprom-1m, 5¢-GAGAATTCGGGTTCGGGAGGAGGAT CC-3¢; ON-EcoRI-prom-2c, 5¢-GAGAATTCCTGGCCCA CTATTTACCCTCC-3¢; ON-hGAPDH-F2, 5¢-TGGGCTA CACTGAGCACCAG-3¢; ON-hGAPDH-R2, 5¢-GGGTGT CGCTGTTGAAGTCA-3¢; ON-GPBP-F1, 5¢-CTGAATCC AGCTTGCGTCG-3¢; ON-GPBP-R1, 5¢-GCAGAGTAGC CACTTGCTCC-3¢ Antibodies All antibodies were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) Plasmid construction To obtain LpromGPBP and LpromPolj, a 772-bp DNA fragment generated by XbaI and EclXI digestion of a 955-bp fragment obtained by PCR from human DNA using ONGPBP-18 m and ON-GPBP-6c (GenBank accession number AF315603) was filled-in and cloned into the HincII site of 5302 F Granero et al pFGH (Nichols Institute, Corning Nichols Institute, San Juan Capistrano, CA, USA) in the orientation of COL4A3 BP or POLK, respectively To generate SpromGPBP and SpromPolj, the 140-bp DNA fragment, generated by PCR using LpromGPBP, ON-XbaG ⁄ Bpro1m and ON-XbaG ⁄ Bpro1c, was digested with XbaI and cloned in pFGH in each of the two orientations Subsequently, SpromGPBP was used to obtain constructs in which NFjBlike and ⁄ or Sp1 binding sites and ⁄ or TATA-like element were selectively deleted (DNFjB and ⁄ or DSp1 and ⁄ or DTATA, respectively) This was accomplished using ONNFjBmut and ⁄ or ON-SP1Del and ⁄ or ON-TATADel and a site-directed mutagenesis approach (Clontech, Mountain View, CA, USA) To obtain the corresponding mutants for transcription in the POLK direction, the reverse orientation was cloned by XbaI digestion and re-ligation To generate human 127-bp pFGH-based constructs, SpromGPBP was digested with BamHI and the resulting DNA fragment cloned into the BamHI site of pFGH in each of the two orientations To obtain the 140-bp mouse homologue constructs, a DNA fragment generated by PCR using ON-PromMouse-F and ON-PromMouse-R and mouse DNA was digested and cloned into the XbaI site of pFGH in each of the two orientations For ribonuclease protection assays, LpromGPBP was digested with ApaI and EclXI and the resulting DNA 503-bp fragment made blunt-ended and cloned into the HincII site of Bluescribe M13+ (Stratagene, La Jolla, CA, USA) to generate BS-ApaI ⁄ EclXI For similar purposes, human DNA was PCR-amplified using ON-polj-2m or ON-polj-3m and ON-GPBP-5c, and the resulting 615- and 337-bp fragments cloned similarly to generate BS-polj2m ⁄ GPBP5c and BS-polj3m ⁄ GPBP5c, respectively All DNA constructs were characterized by nucleotide sequencing RNA purification Total RNA was prepared from cultured cells using TRIREAGENT (Sigma, St Louis, MO, USA) or RNeasy (Qiagen, Hilden, Germany) following manufacturer’s instructions Ribonuclease protection assays BS-ApaI ⁄ EclXI was linearized by EcoRI or HindIII digestion at the polylinker region, and BS-polj3m ⁄ GPBP5c and BS-polj2m ⁄ GPBP5c by digesting the insert with BamHI and EclXI, respectively T3 or T7 ribonucleotide probes representing the antisense of GPBP or pol j mRNAs were obtained using MAXIscriptTM T7 ⁄ T3 in vitro transcription kit (Ambion, Austin, TX, USA) Individual ribonucleotide probes were subject to ribonuclease protection assays using RPAIIITM (Ambion) FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS F Granero et al and total RNA from human cultured hTERT-RPE1 (Clontech) or HEK 293 cells (ATCC # CRL-1573) Promoter fragments These were obtained by PCR using specific oligonucleotides and SpromGPBP or derived mutants as templates Thus, the 140- and 76-bp promoters were obtained using SpromGPBP, ON-NFjB-1m, and ON-TATA-1c or ON-SP1-3c, respectively The140-bpDNFjB and 76-bpDNFjB fragments were obtained using DNFjB mutant, ONNFjBmut, and ON-TATA-1c or ON-SP1-3c, respectively To obtain 76-bpDSp1 we used ON-NFjB-1m, ON-SP1del-c and DSp1 mutant; for 76-bpDNFjB ⁄ DSp1 we used ONNFjBmut, ON-SP1del-c and DNFjB ⁄ Sp1 double mutant To obtain the 26-bp and 26-bpDNFjB promoter fragments, we annealed ON-NFjB-1m and ON-NFjB-1c or ONNFjBmut-2m and ON-NFjBmut-2c, respectively When the DNA was used for protein binding or purification studies, one oligonucleotide was 5¢-end labelled with 32 P or with biotin, respectively PCR For PCR we used Pfu DNA polymerase (Stratagene) or the Expand Long Template PCR System (Roche, Indianapolis, IN, USA) TNF transcriptional induction of GPBP For transient gene expression assays, NIH3T3 cells (1.4 · 105) were seeded in 9.5-cm2 plates, cultured for 14–16 h and transfected for 16–18 h with lg each individual pFGH-derived plasmid and lg pSV-b-galactosidase expression vector (Promega, Madison, WI, USA) using the calcium phosphate precipitation method of the Profection Mammalian Transfection System (Promega) After transfection, cells were rinsed with NaCl ⁄ Pi, fresh medium was added and the levels of human growth hormone in the media and b-galactosidase activity in cell lysates was determined after 48 h following the supplier’s recommendations For some purposes, after transfection the cells were cultured in low serum (0.5%, v ⁄ v) medium for 24 h Fresh low serum medium containing or not containing human recombinant TNFa (10 ngỈmL)1) or TNFb (50 ngỈmL)1) (Sigma) was added and after 48 h, levels of human growth hormone and b-galactosidase were similarly determined Nuclear extract preparation HEK293 cells were grown approximately to 70–80% confluence, culture medium was removed, and fresh culture medium containing 0.5% (v ⁄ v) serum was added and culture continued After 24 h, fresh low serum medium containing or not containing TNF (see above) was added and cultured for another 12–14 h and used for nuclear extract preparation essentially as described previously [31] Real-Time PCR Total RNA (5 lg) from human cultured cells was reversetranscribed with random hexamers using the Ready-To-Go system (GE Healthcare, Chalfont St Giles, UK) following manufacturer’s recommendations, and the mixtures were used for mRNA determinations performing real-time PCR analysis with an SDS 5700 (Applied Biosystems, Foster City, CA, USA) For these purposes, lL of either : 10 dilution of the mixtures for GPBP or : 500 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analysed by the relative quantitation method (DDCt) according to the manufacturer’s recommendations GAPDH was used as an endogenous control to normalize quantification The pair of oligonucleotides used were ON-GPBP-F1 and ON-GPBP-R1 for GPBP; and ON-hGAPDH-F2 and ON-hGAPDH-R2 for GAPDH EMSA and purification of DNA-binding proteins 32 P-labelled or biotin 5¢-modified oligonucleotides were used either to anneal with the corresponding unlabelled complementary synthetic oligonucleotide or to perform PCR with a specific unlabelled oligonucleotide to produce doublestranded labelled DNA, which was further gel purified and used for EMSA (32P) or for nuclear extract protein purification (biotin labelled) following procedures described previously [31] Competitors for EMSA were prepared by annealing ON-NFjB-1m and NFjB-1c (NFjB-L); ON-SP1-3 m and ON-SP1-3c (SP1); ON-TATA-1m and ON-TATA-1c (TATA-L); ON-NFjBmut-2m and ON-NFjBmut-2c (26-bpDNFjB used as nonrelevant DNA) Cell culture and transient gene expression assays Transfection of small interfering RNA (siRNA), chromatin immunoprecipitation (ChIP) and western blot coupled assays Cells were grown in Dulbecco’s modified Eagle’s medium (NIH3T3 and HEK293) or Dulbecco’s modified Eagle’s Ham’s F-12 medium (hTERT-RPE1) containing 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin, and supplemented with 10% (v ⁄ v) of calf (NIH3T3 cells) or fetal bovine (hTERT-RPE1 and HEK 293) serum HEK293 cells were transfected in the absence or in the presence of 50 nm Sp1 or p65 siRNA [32,33] After 24 h, transfection medium was replaced by fresh medium and culture maintained for days Subsequently, cells were cultured for an additional day with low serum (0.5%) and either noninduced or induced with TNFa (10 ngỈmL)1) for FEBS Journal 272 (2005) 5291–5305 ª 2005 FEBS 5303 TNF transcriptional induction of GPBP h, and used for western blot or ChIP assays For western blot purposes, cells were lysed in 10 mm Tris ⁄ HCl (pH 8), 100 mm NaCl, mm EDTA, 1% (v ⁄ v) SDS, 2% (v ⁄ v) Triton X-100, mm phenylmethylsulfonyl fluoride and 10 lgỈmL)1, centrifuged at 20 000 g for 45 and supernatants were analysed to estimate Sp1 or p65 content using specific antibodies For ChIP purposes, cells were treated essentially as previously described [34,35] Pellets containing DNA immunoprecipitates were dissolved in 60 lL H2O and lL were subjected to PCR using ON-EcoRI-prom-1m and ON-EcoRI-prom-2c (30 cycles each consisting of 95 °C for 45 s; 60 °C for 30 s; 72 °C for 45 s with initial heating at 95 °C for and a final extension step at 72 °C) to verify the presence of the 140-bp promoter The DNA in the supernatants of nonrelevant immunoprecipitations was ethanol precipitated, similarly hydrated and further diluted : 50 in H2O prior to being used (2 lL) in PCR procedures as DNA input samples Acknowledgements This work was supported by grants SAF97 ⁄ 0065, SAF2000 ⁄ 0047, SAF2001 ⁄ 0453 and SAF2003-09772 of the Plan Nacional de I + D, and grant 98 ⁄ 102-00 of ´ Fundacion ‘La Caixa’ (Spain) (to J.S), and the fellowship program of the F.V.I.B in collaboration with BioStratum Inc and Iberdiagnosis S.A References Raya A, Revert F, Navarro S & Saus J (1999) Characterization of a novel type of serine ⁄ threonine kinase that specifically phosphorylates the human Goodpasture antigen J Biol Chem 274, 12642–12649 Raya A, Revert-Ros F, Martinez-Martinez P, Navarro S, ´ Rosello E, Vieites B et al (2000) Goodpasture antigenbinding protein, the kinase that phosphorylates the Goodpasture antigen, is an alternatively spliced variant implicated 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ON-XbaG ⁄ Bpro1m, 5¢-GA CTCTAGAGGGTTCGGGAGGAGGATCCCG-3¢; ONXbaG ⁄ Bpro1c, 5¢-GACTCTAGACTGGCCCACTATTTA CCCTCC-3¢; ON-GPBP-5c, 5¢-CTCGATGCCAATTTCAA ATAGGGAA-3¢; ON-polj-2m, 5¢-GACAAGCCGCCCTG GAAAGCAGGCCC-3¢;... keratinocyte maturation and autoimmune pathogenesis Experimental procedures Synthetic oligonucleotides ON-GPBP-6c, 5¢-CTCGCTCGCCCAGGGAAGGAAAA GGGAAAAGAAGGGA-3¢; ON-GPBP-18 m, 5¢-GGCAT GGTTAACGTGGTTCTC-3¢;... 5¢-GCAGCACAGC TGCATCCCTACCCCGCCCTCTC-3¢; ON-SP1Del, 5¢-CG CCGGGAGGGGGACGTAGTGGGGGAGAAT-3¢; ONNFjBmut, 5¢-TCTAGAGGGTTCGGGAGAAGGCTCGG CGTGTCG-3¢; ON-TATADel, 5¢-CAGGGGAGGGGAG GGGTGGGCCAGTCTAGA-3¢;