A duplicated gene in the breakpoint regions of the 7q11.23 Williams-Beuren syndrome deletion encodes the initiator binding protein TFII-I and BAP-135, a phosphorylation target of BTK
Luis A. Pérez Jurado1,2,3, Yu-Ker Wang4, Risa Peoples3, Antonio Coloma2, Jesús Cruces2, Uta Francke3,4,*
1Unidad de Genética, Hospital Niño Jesús and 2Departimento de Bioquímica, Facultad de Medicina, Universidad Autónoma, Madrid, Spain and 3Department of Genetics and 4Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, CA 94305, USA
Received October 6, 1997;Revised and Accepted December 8, 1997
Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder with multisystemic manifestations caused by heterozygosity for a partial deletion of chromosome band 7q11.23. The breakpoints cluster within regions located ~1 cM either side of the elastin (ELN) locus. We have characterized a duplicated region near the common deletion breakpoints, which includes a transcribed gene. The centromeric (C) and telomeric (T) copies are almost identical in the duplicated 3[prime] portions but diverge at their 5[prime]-ends. C-specific 4.3 kb mRNA and T-specific 5.4 kb mRNA are widely expressed in embryonic and adult tissues. The telomeric gene gives rise to several alternatively spliced forms and is deleted in all WBS individuals who have documented ELN deletions. Database searches revealed that this gene encodes BAP-135, a protein phosphorylated by Bruton's tyrosine kinase in B cells, as well as the multifunctional transcription factor TFII-I, hence the gene name GTF2I. The centromeric gene is not deleted in WBS and appears to be a partially truncated expressed pseudogene with no protein product (gene name GTF2IP1). Both loci map to different genomic clone contigs that also contain other deleted and non-deleted loci. A probe from the shared region recognizes a >3 Mb NotI junction fragment that is unique to individuals with the WBS deletion. Therefore, the duplicated region containing GTF2I and GTF2IP1 respectively is located close to the deletion breakpoints and may predispose to unequal meiotic recombination between chromosome 7 homologs and/or to intrachromosomal rearrangements. Hemizygosity for GTF2I may also contribute to the WBS phenotype.
Williams-Beuren syndrome (WBS) is a multisystemic neurodevelopmental disorder characterized by distinctive facial features, mental disability with unique cognitive and personality profiles, supravalvar aortic and other vascular stenoses, growth retardation and occasional infantile hypercalcemia, caused by haploinsufficiency for genes in the deletion at chromosome band 7q11.23 (1-4). Consistently included within the deleted interval are the loci encoding elastin (ELN), replication factor C subunit 2 (RFC2), LIM kinase-1 (LIMK1), the wnt receptor Drosophilafrizzled homolog FZD3, the transcript WBSCR1 and syntaxin1A (STX1A), as well as other ESTs and transcription units of unknown function (4-11). This common deletion is present in the majority of WBS individuals studied, with only 4-5% of clinically diagnosed WBS cases not deleted for any of the available markers (5,6,12-14). Rarely, affected parents and children have been reported, but the great majority of WBS cases are sporadic, with an estimated incidence of 1 in 20 000 live births, indicating a very high mutation rate (~0.5 × 10-4) (3,15,16).
Little is known about the mechanisms predisposing to the frequent mutational events. Genetic and physical mapping data are consistent with clustering of common deletion breakpoints in relatively small genomic regions on both sides of the ELN locus (13,14). Meiotic recombination between polymorphic markers proximal and distal to the deleted interval was documented in most informative families, suggesting unequal crossing over between misaligned homologous regions as the most likely mutational mechanism (17,18). In one study no evidence for unequal interchromosomal exchange was found in some cases, indicating that intrachromosomal meiotic or mitotic rearrangements can also be responsible. The WBS deletions may be of paternal or maternal origin, with a slight but not significant increase in maternally derived deletions (5,13,17,18). The discovery of a dinucleotide repeat polymorphism that represents more than one locus (D7S489) in 7q11.23 suggested the presence of low copy number repeats in the region (13,14).
We report here the characterization of partially duplicated sequences from the common breakpoint regions that include a protein coding gene with multiple alternatively spliced potential products. The shortest splice product encodes BAP-135, a protein identified as a target for phosphorylation by Bruton's tyrosine kinase in B cells (19). The sequence is also identical to the recently published cDNAs for a multifunctional DNA binding component of transcription complexes, named SPIN (20) or TFII-I (21). While the genomic duplication may represent a hotspot for the unequal recombination events leading to the WBS deletions, hemizygosity for this ubiquitously expressed transcription factor may contribute to the WBS phenotype.
As we have previously reported, the breakpoints leading to the WBS deletions cluster in genomic regions flanking the elastin locus. The commonly deleted interval is defined by loci D7S489B (centromeric) and D7S1870 (telomeric), that recombine in 2% of meioses (13). Yeast artificial chromosomes (YACs) containing these loci were identified by PCR typing and assigned to either side of the deletion by their sequence-tagged site (STS) content (Fig. 1). Inter-Alu PCR probe hybridizations and restriction mapping revealed high sequence homology between YAC clones from both sides of the deletion interval, suggesting a genomic duplication. An expressed sequence tag (EST), IB291 (22), was mapped to YAC 763H7 in the fifth release of the Whitehead Institute-MIT human genome database (23). By PCR analysis of a somatic cell hybrid panel we confirmed that IB291 maps to the long arm of chromosome 7 (not shown). Southern hybridization of an IB291 probe to somatic hybrid cell lines containing either a normal chromosome 7 or chromosome 7 with a WBS interstitial deletion on a Chinese hamster cell background (7) generated a signal of reduced intensity in the hybrid cell lines that contained the deleted chromosome 7 (not shown). The observed gene dosage difference indicates that IB291 recognizes at least two loci on chromosome 7 and that one of them is lost in the WBS deletion. By PCR and hybridization studies, IB291 was assigned to several YACs located at either side of the ELN locus, confirming its duplicated nature (Fig. 1). Each of these YAC groups also contains a different D7S489 locus that is, therefore, likely to be a part of the genomic duplication. Both YAC contigs contain deletion breakpoints, as previously shown by PCR typing of polymorphic markers in a series of WBS individuals (13).
Figure 1. Physical map of the WBS deletion and flanking regions in 7q11.23 (not to scale). Two YAC contigs and corresponding STS markers encompass the deletion breakpoints on both sides. YAC identifiers are those of the original sources, CEPH* (56) and chromosome 7-specific** (57) YAC libraries. Single copy gene loci within the deletion are shown in italics. The estimated extent of the duplicated region is represented by hatched boxes. EST IB291 was mapped to both centromeric and telomeric YACs near the deletion breakpoints. STS2 and STS3 are described in the text and are more precisely mapped in Figure 2a.
The cDNA clone containing EST IB291 (22) (GenBank accession no. T03439) was obtained from the ATCC and sequenced. Sequence comparisons using the BLAST software (24) revealed sequence identity with many additional ESTs from dbEST. By sequence walking and computer assembling of multiple EST sequences (25), two composite cDNA variants diverging in the 5[prime]-region were identified (Fig. 2a). IMAGE clone 147504 (GenBank accession nos. R81199 and R81308), containing the longest 5[prime]-region, was purchased from Research Genetics and its full sequence verified. STSs specific for each divergent 5[prime]-region (STS2 and STS3; Fig 2a) were developed and used to map each copy to either the centromeric (GTF2IP1) or the telomeric YAC clones (GTF2I) (Fig. 2b). Sequence comparisons indicate that the GTF2IP1 and GTF2I cDNAs share 3.2 kb of their 3[prime]-regions, while the 5[prime]-regions of each gene are unique.
Figure 2 (a) Characterization of two highly related genes from the duplicated region, now named GTF2I and GTF2IP1. A contig was assembled of overlapping EST sequences, shown with their dbEST identifiers. GTF2I (telomeric) and GTF2IP1 (centromeric) are two cDNAs that diverge in their 5[prime]-regions. The part unique to GTF2I is shaded and the unique portion of GTF2IP1 is hatched. Location and direction of PCR primers developed from cDNA sequences are indicated by arrows.
Vertical bars between primers denote the presence of introns at STS3 and STS4, but not at STS1 and STS2. Probes used for hybridization are the GTF2I-specific probe, detecting only the larger telomeric gene, and the cDNA clone IB291, detecting both genes. (b) Mapping of the GTF2I and GTF2IP1 loci with respect to the WBS deletion. Three different STSs were studied in Chinese hamster × human somatic cell hybrid lines, containing either the deleted (D) or non-deleted (N) chromosome 7, in Chinese hamster (H) control cells and in two YAC clones, containing either the centromeric (CY) or the telomeric (TY) deletion breakpoints. The results reveal the existence of at least two copies of the 3[prime]-region (STS1), each located in different YACs. STS2, derived from the 5[prime]-exon specific for GTF2IP1, is located in the centromeric YAC and outside the deletion, as it is present in both hybrid cell lines. STS3, derived from the 5[prime]-region specific for GTF2I, is located in the telomeric YAC and is deleted from the D hybrid cell line. No amplification was observed in hamster DNA for any of the STSs.
Northern blot analysis with the 1.3 kb IB291 cDNA insert as probe revealed two transcripts of ~4.3 and 5.4 kb in all tissues tested (Fig. 3a, left). Hybridization with a 840 bp cDNA probe generated by PCR from the GTF2I-specific 5[prime]-region identified the 5.4 kb transcript only (Fig. 3a, right). Thus, GTF2I and GTF2IP1 seem to be expressed as different sized mRNAs.
Figure 3 (a) Northern blot hybridization. With the 3[prime] probe (IB291) two transcripts of 4.3 and 5.4 kb were detected in all adult human tissues (left). A probe from the 5[prime]-end of the GTF2I gene hybridized only to the 5.4 kb transcript (right). The filters were re-hybridized with [beta]-actin cDNA as control. (b) RT-PCR analysis of GTF2I and GTF2IP1 transcripts in embryonic and adult tissues. With forward primers A (GTF2IP1-specific) and C (GTF2I-specific) and reverse primer M (common to both cDNAs) the existence of both types of mRNAs was confirmed in all tissues tested. Alternative splicing of GTF2I-specific products was observed: band 1 contained both exons A and B and was most evident in brain and lymphoblasts, the co-migrating bands 2 and 3 contained either exon A or B respectively, by sequencing, and band 4 lacked both exons. Only a single amplification product corresponding to GTF2IP1 mRNA was obtained from all tissues. (c) Mapping of GTF2I and GTF2IP1 transcripts with respect to the WBS deletion by RT-PCR analysis of somatic cell hybrid lines. Primers A+M amplified the GTF2IP1-specific transcript from the deleted (D) and non-deleted (N) chromosomes 7, while GTF2I mRNAs were detected only in the hybrid cell line with the non-deleted (N) chromosome 7 with the gene-specific primer pairs C+M and F+D. Both types of RT-PCR products were amplified from brain tissue (B) and no amplification was observed from hamster RNA (H) or in control samples lacking RNA (-).
To further characterize the gene copy-specific transcripts, we used primers from the differential 5[prime]-regions (primer A from GTF2IP1 and primer C from GTF2I) (Fig. 2a) along with different reverse primers from the duplicated region to PCR amplify and sequence gene copy-specific cDNAs. By using a fetal brain cDNA library or reverse transcribed RNA as templates, a total sequence of 4.5 kb was obtained for GTF2I and of 3.4 kb for GTF2IP1. Only three nucleotide changes were observed in GTF2IP1 cDNA compared with GTF2I cDNA in the 3.2 kb duplicated region: C1487T, G2217A and C3130T (GTF2I nucleotide numbers as in Fig. 4a). Therefore, these regions have 99.9% nucleotide identity within the cDNA sequence.
Figure 4 (a) Nucleotide sequence and predicted protein product of the longest GTF2I cDNA. Regions that are alternatively spliced out are shaded. The putative nuclear targeting signals are underlined, the putative RNP RNA binding domain is double underlined and the two tyrosine phosphorylation sites are boxed. Potential polyadenylation signals in the 3[prime]-untranslated region are underlined. The vertical arrows indicate the exon-intron junctions. The start of the duplicated region that is also present in GTF2IP1 cDNA is indicated by a horizontal arrow. The potential initiation codon of the GTF2IP1 ORF is labeled with an asterisk and the single amino acid difference with respect to GTF2I (R634H substitution) is indicated. (b) Amino acid sequence alignment of a region from a mutator transposase (IsRm3 from Rhizobium meliloti) (top) and the C-terminal portions of the six GTF2I repeats as identified by BLOCK motif search (31). Identical or similar residues are highlighted. In the putative consensus an amino acid code letter indicates identity of the transposase residue with two or more of the GTF2I repeats. Conserved substitutions include: U, bulky hydrophobic residue (I, L, V, M, F, Y); O, small hydroxyl residue (S, T); $, positively charged basic residue (K, R). (c) Nucleotide sequence of the 5[prime] GTF2IP1-specific region. The in-frame stop codon is underlined.
Reverse transcriptase (RT)-PCR analysis using primers C and M revealed four mRNA variants of GTF2I. Sequence analysis revealed that the different mRNAs resulted from alternative splicing of two exons located upstream of the duplicated region (Figs 3b and 5). The longest GTF2I transcript (band 1) is strongly present in fetal brain (not shown), weakly in adult brain, muscle and lymphoblasts and is almost undetectable in other adult tissues, while the other variant transcripts are equally expressed in all adult tissues (Fig. 3b). With different primer combinations no variation in RT-PCR products was obtained for other regions of the available GTF2I cDNA. With the GTF2IP1-specific primer A and different reverse primers no alternative splicing products were seen, only a single product being expressed in most tissues (Fig. 3b). RT-PCR analysis of RNA from somatic hybrid cell lines containing the deleted (D) or non-deleted (N) chromosome 7 confirmed that GTF2IP1 is not deleted (primer pair A+M) while GTF2I is deleted (primer pairs C+M and F+D) (Fig. 3c).
Figure 5. Comparative genomic structures of GTF2IP1 (top) and GTF2I (bottom). Duplicated exons are represented as filled boxes, the GTF2IP1-specific exon as a hatched box and GTF2I-specific exons as open boxes. Intron sizes in the duplicated region are drawn to approximate the 30 kb scale, but exon sizes are enlarged to show their sizes relative to each other. The positions of the three nucleotide differences in the coding sequences of the duplicated region are marked by arrows. In the GTF2I-specific region question marks within introns indicate unknown size and a question mark within an exon means that an additional intron may exist. Exons A and B are alternatively spliced out, generating four different types of GTF2I transcripts. A single GTF2I-specific cosmid contained D7S1870, commonly deleted in WBS patients (32), as well as D7S2714 (sequence-tagged sites developed at the Stanford Human Genome Center, available on line at http://shgc-www.stanford.edu), indicating that these STS markers are within introns of GTF2I.
To establish the genomic structure of each of the duplicated loci, we identified several cosmid clones by hybridizing chromosome 7-specific cosmid library filters with two GTF2I probes (26). PCR analysis with locus-specific primers allowed us to assign the positive cosmids to either the centromeric (GTF2IP1) or the telomeric (GTF2I) locus. Exon-intron boundaries were identified by comparison of PCR amplifications from cDNA and genomic DNA with various combinations of primers designed from the cDNA sequence (primer sequences available upon request). The duplicated region contains 21 exons and spans ~30 kb of genomic DNA (Fig. 5). Exon and intron sizes are identical in GTF2IP1 and GTF2I and the sequenced intron-exon junctions follow the canonical AG-GT rules in both genes. Only one exon that is specific for the 5[prime]-region of GTF2IP1 has been identified. The genomic structure of the non-duplicated 5[prime]-region of GTF2I has been determined by primer walking because of the large size of several introns. It contains at least 12 exons and spans >70 kb of genomic DNA.
PCR amplification and Southern blot hybridization of genomic DNAs with several GTF2I primers and probes revealed the presence of cross-hybridizing signals corresponding to related sequences outside the WBS deletion. During BLAST searches we have identified ESTs with some sequence divergence from GTF2I (90-95% identity), which may correspond to these additional copies or related genes.Preliminary data reveal that copies of related genes exist on chromosome 7 as well as on several other chromosomes. Based on analysis of genomic DNA from several individuals we believe that the number of additional copies may be variable in the population (data not shown).
The longest GTF2I cDNA has a 2994 nt (nt 316-3309)open reading frame that predicts a 998 amino acid protein (predicted molecular mass 112.4 kDa) (Fig. 4a). The putative initiator methionine codon is within a context favorable for translation initiation (27). The discrepancy between the cloned cDNA (4.5 kb) and the transcript size determined by northern blot (5.4 kb) must be due to additional unidentified exons in the 5[prime]-untranslated region. Attempts to extend the cDNA further by use of 5[prime]-RACE were unsuccessful.
Alternative splicing of two exons immediately upstream of the duplicated region maintains the reading frame in all combinations and predicts the generation of four protein variants. The second and shortest mRNA variant, lacking exons A and B (Fig. 5), encodes BAP-135, a recently identified 957 amino acid novel protein (observed molecular mass 135 kDa, predicted molecular mass 107.8 kDa), which is a target for phosphorylation by Bruton's tyrosine kinase (Btk) in response to B cell receptor engagement (19). It has been proposed that the discrepancy between the calculated and observed molecular masses may be due to the high percentage of acidic residues. In vitro expression of a cloned cDNA that used the predicted initiation codon as the initiator methionine resulted in a protein of the same molecular mass as endogenous BAP-135, suggesting that the proposed initiation site is the one used in vivo (19). Five amino acid differences between the published BAP-135 sequence and ours are likely due to sequencing errors, as our GTF2I sequence is identical to that for SPIN (20; GenBank accession no. Y14946).A third mRNA species (lacking exon B) encodes a 977 amino acid protein (predicted molecular mass 110.1 kDa) and a fourth mRNA (lacking exon A) encodes a 978 amino acid protein (predicted molecular mass 110.2 kDa). Interestingly, a protein of apparent 140 kDa was also found as a target for Btk phosphorylation and also reacts with antibodies raised against BAP-135 (19). This protein is likely to be the translation product of one of the larger GTF2I mRNA species produced by alternative splicing in lymphoblasts. Of note, there is a significant difference in the isoelectric points between the predicted GTF2I protein isoforms, dropping from 8.23 for BAP-135 to 6.09 for the largest 998 amino acid variant that includes both exons A and B.
Prediction of known sequence motifs using the PSORT program (28) suggests two putative nuclear targeting signals in all four potential GTF2I-encoded proteins. A PROSITE search revealed an RNA binding motif near the N-terminus as well as potential targets for tyrosine kinase phosphorylation (29; Fig. 4a). There are six amino acid repeats within the protein sequence from which a consensus sequence has been derived (19). With the BLOCKS algorithm (30) we detected a similarity between the six polypeptide repeats of the GTF2I gene and a putative transposase of the Mutator group of bacterial insertion sequences and transposable elements in maize (31; Fig. 4b).
The shorter GTF2IP1-specific cDNA has an in-frame stop codon within the unique 5[prime]-region (Fig. 4c). The three nucleotide changes in the duplicated region, compared with GTF2I, cause an additional in-frame termination codon at amino acid 391 of the GTF2I sequence (Fig. 4a), a missense substitution (R634H) and a silent change in the wobble position. There is a potential translation initiation codon downstream of the second in-frame stop codon followed by an open reading frame that would encode a 530 amino acid protein almost identical to the C-terminus of GTF2I with the single R634H substitution (Fig. 4a). This potential initiator codon has a poor Kozak consensus (27). In addition, an antibody raised against a BAP-135 peptide that should be present in this hypothetical GTF2IP1-encoded protein product failed to detect any smaller proteins (19). This information suggests that GTF2IP1 mRNA, despite being highly expressed and processed, does not get translated into a mature protein, at least not in the tissues examined.
PCR typing of several GTF2I-specific STSs in hybrid cells with chromosome 7 bearing the typical WBS deletion showed lack of amplification products, while GTF2IP1-specific products and fragments common to both loci did amplify (Fig. 2b). Furthermore, ICRF cosmid clone c113L1513, spanning several internal GTF2I exons (Fig. 5), was found to contain two STSs, D7S2714 (sequence-tagged sites developed at the Stanford Human Genome Center, available on line at http://shgc-www.stanford.edu) and D7S1870, which must, therefore, lie within introns of GTF2I. Since D7S1870 is usually deleted in WS patients (13,14,32), this region of GTF2I falls within the commonly deleted interval.
The few known sequence differences provide tools for further deletion analysis. The G[rarr]A change in GTF2IP1 (R634H) causes the absence of a SacII/BstUI restriction site that is present in GTF2I. In the adjacent intron there is an RsaI restriction site in GTF2IP1 that is absent in GTF2I. Using primers flanking this region for PCR and subsequent restriction digestion of the amplified products with BstUI and RsaI revealed a dosage reduction of the GTF2I-specific fragments in most WBS samples with respect to controls (Fig. 6). Two WBS individuals, known to be deleted at D7S1870 (13), did not show dosage reduction. This could be due to the presence of additional related sequences which also amplify and interfere with the analysis. Alternatively, the telomeric deletion breakpoints in these individuals could lie between D7S1870 and the region amplified by PCR near R638H, which are at least 20 kb apart (Fig. 5).
Figure 6. Deletion analysis of the GTF2I and GTF2IP1 loci in WBS patients and controls. PCR products obtained with primers for STS4, which contain exon and intron sequences of the duplicated region, were cleaved with BstUI and RsaI and were separated in a 4% agarose gel. Lanes M contain pUC plasmid DNA digested with HpaII as molecular size marker. Lane 1 contains PCR products from a GTF2I-specific cosmid and lane 2 contains PCR products from a GTF2IP1-specific cosmid. Lanes 3, 5, 7 and 9 represent WBS individuals and lanes 4, 6, 8 and 10 independent normal controls. A dosage reduction is evident for the GTF2I-specific fragments (arrows) in lanes 3, 5 and 7 compared with controls. The patient in lane 9 appears to have a normal dosage ratio in spite of being deleted at locus D7S1870.
Following digestion with NotI and pulsed field gel electrophoresis, novel deletion junction fragments >3 Mbp in size were detected with the IB291 cDNA probe in the hybrid cell lines with the WBS deletion chromosome 7, as well as in four WBS samples. The corresponding fragments in normal control samples and somatic cell hybrids retaining the non-deleted chromosome 7 were ~2.5 Mbp in size (data not shown). Frequently cutting enzymes and other rarely cutting enzymes, however, did not generate novel junction fragments on Southern blots probed with GTF2I cDNA, although reduced intensity of the fragments compared with controls suggested a dosage effect. We interpret these results to indicate that the breakpoints leading to the WBS deletions occur within or close to the GTF2I and GTF2IP1 loci. However, because of the extremely high sequence identity, most restriction sites may be identical in both genes. If recombination events leading to WBS deletions occur between aligned highly similar but non-homologous sequences, most of the resulting junction fragments may be identical in size to the original fragments and, therefore, impossible to detect.
More than 95% of clinically defined WBS patients have de novo deletions, quite homogeneous in size, at chromosomal band 7q11.23 (13,14). We have identified a genomic duplication flanking the commonly deleted interval. The duplication includes ~30 kb of the 3[prime]-region of a transcribed gene, called GTF2I (telomeric copy) and GTF2IP1 (centromeric copy). The duplicated region is >30 kb, however, and encompasses D7S489 loci as well as genes related to EST IB1445, belonging to the PMS2 mismatch repair gene family (33), and other as yet uncharacterized sequences. The very high conservation (99.9%) of the coding sequences of GTF2I and GTF2IP1, as well as the preservation of restriction fragment patterns at both loci, indicate that the duplication is of recent evolutionary origin. Indeed, as reported elsewhere, there is a single locus, corresponding to GTF2I, in mouse (named Gtf2i) and probes from this gene also detect single transcripts in rat (34).
The duplicated genes in humans differ in their products and deduced functions. GTF2I, at the telomeric repeat, is transcribed and alternatively spliced into several mRNA species with long ORFs that encode various protein isoforms, one of which is identical to BAP-135 (19), SPIN (20) and TFII-I (21). GTF2IP1, in the centromeric repeat, is transcribed into a shorter mRNA which is apparently not translated. We conclude that the telomeric repeat contains a functional gene and the centromeric repeat a truncated pseudogene.
De novo germline deletions causing WBS occur at an approximate rate of 0.5 × 10-4, however, there is little information addressing the mechanisms of these rearrangements. Haplotypes recombinant for segments flanking the deleted region have been demonstrated in most WBS chromosomes studied, indicating that these deletions result from unequal crossing over between the chromosome 7 homologs during gametogenesis (17,18). Recombination events between misaligned repetitive elements have been observed at the deletion and duplication breakpoints in a variety of DNA rearrangements resulting in human genetic disorders. Low copy number repeat sequences have been found dispersed within some chromosomal regions that are subject to spontaneous deletions, such as Xp22.3 (Kallman syndrome; 35), 22q11 (DiGeorge/velo-cardio-facial syndromes; 36), 15q11-13 (Prader-Willi/Angelman syndromes; 37,38) and 5q13 (spinal muscular atrophy; 36). Both the repetitive nature of these sequences and their clustering within a relatively small interval provide a mechanism for aberrant recombination events and, thus, the high frequency of interstitial deletions in these regions. In addition, there are large rearrangements between repeats on the X chromosome leading to steroid sulfatase gene deletions (39) and reciprocal duplication and deletion products of unequal crossing over events on chromosomal band 17p11.2 that are responsible respectively for Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) (40,41). In these two cases a precise mechanism for homologous unequal recombination has been found to occur between misaligned repeats that are duplicated in the regions flanking the rearrangements (42). After submission of this manuscript a similar mechanism has also been reported for deletion formation in Smith-Magenis syndrome (43). Furthermore, unequal crossing over at homologous genes has led to variation in the copy number of green pigment genes in humans (44) and X-linked color blindness is due to a similar mechanism resulting in complete deletion of the pigment genes or recombination leading to hybrid red and green genes (45).
Our data suggest a similar situation for WBS deletions. Typing of multiple polymorphic markers has previously indicated that deletion breakpoints cluster in regions which, as shown here, are part of a recent evolutionary genomic duplication. A probe from this duplicated region detected large junction fragments of similar size with long range restriction enzymes in five unrelated WBS individuals. Therefore, unequal recombination appeared to occur in a similar manner in multiple unrelated WBS chromosomes, further supporting the idea of a common mechanism for the deletions.
GTF2I is deleted in all informative WBS patients with a documented ELN deletion at 7q11.23. Therefore, these patients are hemizygous for the functional copy of the gene and are expected to produce half-normal dosage of its protein product(s). BAP-135, a GTF2I-encoded protein, has been identified as a target of phosphorylation by Btk during B cell receptor engagement (19). It occupies a position immediately downstream of Btk in the B cell receptor activating pathway. Since Btk mutations cause X-linked agammaglobulinemia, mutations or deletions in BAP-135 have been speculated to be associated with a similar immunodeficiency phenotype in the homozygous state (19). However, B cell function appears to be within normal limits in WBS individuals heterozygous for deletions of GTF2I/BAP-135. In addition, the expression pattern of GTF2I is much broader than that of Btk and includes multiple fetal tissues. Therefore, its functional role is likely to extend beyond signaling in B cells. As suggested, the BAP-135 tyrosine phosphorylation signals could also be targets for tyrosine kinases other than Btk whose expression is B cell specific (19).While Btk is a cytoplasmic protein and Btk-BAP-135 interactions would be expected to occur in the cytoplasm, the presence of putative nuclear localization signals in the GTF2I sequence suggested a function in the nucleus.
Also intriguing is a weak similarity of the six amino acid repeats in GTF2I/BAP-135 to putative transposases from plants and bacteria (31; http://www-leland.stanford.edu/~eisen/Mutator/Mutator.html). Transposases are implicated in some of the mechanisms of meiotic recombination, such as reciprocal breakage, exchange of DNA segments and rejoining of chromatids (46). Interestingly, a mariner transposon-like element is part of the duplicated region that is the recombination hotspot leading to duplication, in CMT1A, and deletion, in HNPP, of a 1.5 Mb region of 17p12-p11.2 (47). This mariner element contains a transposase-like sequence with similarity to insect transposases. This analogyinitially led us to speculate that GTF2I may have transposase-like function and that the duplicated genomic region could be a target for its own protein product which could, therefore, contribute to the unequal recombination events that cause the WBS deletions.
After our manuscript was submitted other functions of this gene were reported independently by two groups. Grueneberg et al. (20) cloned and functionally characterized the cDNA for a multifunctional DNA binding protein, termed SPIN, that binds to the c-fos promoter and interacts with serum response factor and with the homeobox protein Phox1. Studying a quite different experimental system, Roy et al. (21) succeeded in cloning the transcription factor TFII-I that binds core promoters at the pyrimidine-rich initiator element (inr) as well as at the upstream E box and is an essential component of a transcription factor complex. Both human SPIN and TFII-I cDNA sequences were found to be almost the same as BAP-135 and correspond to the shortest of the four different splice products of GTF2I that we identified.
In summary, we have characterized a genomic repeat which includes a duplicated gene, named GTF2I and GTF2IP1, flanking the interval at 7q11.23 commonly deleted in WBS. Unequal crossing over between chromosomes or chromatids paired at these misaligned repeats appears to be the major mechanism responsible for de novo WBS deletions. The recombination occurs in a similar manner in multiple unrelated individuals, resulting in a junction fragment that lacks the telomeric (and functional) locus GTF2I.Therefore, hemizygosity for GTF2I, that encodes a multifunctional member of a eukaryotic transcription factor complex and is tyrosine phosphorylated by Btk, is a consistent feature in WBS and haploinsufficiency for this protein may contribute to the WBS phenotype. There is a growing list of human multisystem developmental disorders that are caused by haploinsufficiency for transcriptional co-activators or DNA binding proteins with potential function as transcription factors. For example, Rubinstein-Taybi syndrome, characterized by broad thumbs and great toes, mental retardation, agenesis of corpus callosum, typical facial features, eye, skin, skeletal and cardiovascular abnormalities, is caused by deletion or disruption of the transcriptional co-activator CREB binding protein (48,49). Haploinsufficiency for the paired-box DNA binding protein PAX6 results in eye malformations, including aniridia (50), while deletion or inactivating mutations of PAX3 give rise to a form of Waardenburg syndrome with pigmentary disturbance and hearing loss (51). The GLI3 gene, a member of GLI-Kruppel zinc finger gene family that represents a potential sequence-specific DNA binding transcription factor, is deleted in patients with Greig cephalopolysyndactyly syndrome (52), while frameshift mutations cause Pallister-Hall syndrome (53). Therefore, when considering the contributions to the phenotype of candidate genes identified in the 2 Mb WBS deletion those with potential functions as transcription factors/co-activators, such as GTF2I, are likely to be dosage sensitive and should be taken seriously.
Criteria for the clinical diagnosis of WBS in the five individuals included in this study have been reported elsewhere (13). Genomic DNA was isolated from peripheral blood lymphocyte samples or from Epstein-Barr virus-transformed lymphoblastoid cell lines by standard techniques and RNA was isolated using RNA STAT-60 (Tel-Test'B Inc.) as described by the manufacturer.
Initial assignment of IB291 was performed by PCR amplification of our panel of rodent × human somatic cell hybrids whose content of human chromosomes has been previously documented by cytogenetic and molecular methods (54). Somatic cell hybrid lines in which the normal chromosome 7 has been separated from the chromosome 7 carrying the interstitial deletion of a WBS patient on a Chinese hamster cell background have been reported (7). Deletion mapping was performed by PCR typing or genomic Southern hybridization using DNA from the hybrid cell lines.
EST sequences were tested for similarity to known sequences in GenBank using the BLAST software (24). cDNA clones were purchased from Research Genetics (clone 147504) and ATCC (no. 86072, containing EST IB291). A 5[prime] GTF2I-specific cDNA probe was generated by PCR from clone 147504 with primers F (5[prime]-ATGTCCACCCTCCCCGTTGA-3[prime]) and D (5[prime]-GGTGGCTTCCTTGAATGTTA-3[prime]). Larger cDNAs were obtained by PCR-based screening of a human fetal brain cDNA library cloned in [lambda]ZAPII (Stratagene).
A chromosome 7-specific cosmid library spotted on high density filters (26) was kindly provided by Hans Lehrach (Resource Center, Max Planck Institute for Molecular Genetics, Berlin, Germany). Library screening was performed with random hexamer-labeled cDNA probes and positive clones were obtained from the same source. Multiple oligonucleotide primers were designed based on the cDNA sequence and were used for PCR of cDNA and genomic DNA. Comparison of cDNA and genomic sequences permitted estimation of intron sizes and identification of intron-exon junctions and exon boundaries.
STSs were developed to identify and distinguish GTF2I and GTF2IP1 by PCR (Fig. 2). STS1 (both genes) = IB291 (22,23); STS2 (GTF2IP1-specific), 5[prime]-CGGACGATGGAACTCCACAT-3[prime] and 5[prime]-GACAGGAAGGCAAGTTTGAT-3[prime] to amplify 108 bp; STS3 (GTF2I-specific), 5[prime]-AGGCAAATCCACAGTGGTAC-3[prime] and 5[prime]-CTACATGCTTCTTTGGCTCT-3[prime] to amplify 380 bp; STS4 (both genes), 5[prime]-GCTCAAGCTCTTGGACTCAC-3[prime] and 5[prime]-CAGGAGGCAAGTAGGAAATA-3[prime] to amplify 1.2 kb.
Restriction analysis was performed by digestion of 10 µl PCR-amplified products (STS4) from cosmid or genomic WBS and control DNAs with RsaI and BstUI (NEB) according to the manufacturers. The resulting fragments were analyzed by ethidium bromide staining following electrophoresis in 3% agarose gels.
Sequencing of cloned DNA was performed on an ABI prism 377 sequencer (Perkin Elmer). Double-strand sequencing of PCR-amplified products was performed using a PCR sequencing kit (US Biochemical). The cDNA sequences have been deposited in GenBank: GTF2I, accession no. AF035737; GTF2IP1 accession no. AF036613.Protein sequence comparisons were carried out with the BLASTP (24), BEAUTY (55) and PROSITE (29) programs and the BLOCKS database version 9.1 (30). Prediction of protein motifs was with PSORT (28).
A northern blot filter containing poly(A)+ RNA from multiple human tissues was purchased from Clontech and probed according to the manufacturer's instructions. Hybridization probes were generated by PCR from plasmid clones or by RT-PCR from tissues. PCR products were gel purified and radiolabeled with random primers (Pharmacia). The probes were hybridized overnight at 65°C in Church buffer and washed at a final stringency of 0.1× SSC, 0.1% SDS at 65°C.
For RT-PCR studies tissues were obtained from an adult female cadaver, according to an IRB approved human subjects in research protocol and snap frozen in liquid nitrogen. Tissues were dispersed using a polytron homogenizer directly into RNA STAT-60 (Tel-Test'B Inc.) and total RNA was extracted as per the manufacturer. One microgram of total RNA was used for first strand cDNA synthesis, with 200 U SuperScriptII reverse transcriptase (Gibco BRL) and random hexamers, in 20 µl total volume and incubated for 60 min at 42°C. One microliter of the reaction was used for 35 cycles of PCR at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min and a final extension at 72°C for 7 min. Primer sequences were: GTF2IP1-specific, A, 5[prime]-CGGACGATGGAACTCCACA-3[prime]; GTF2I-specific, C, 5[prime]-CCCCATCAAAGTGAAAACTG-3[prime]; common to both genes, M, 5[prime]-AGTTGAACTCCCTCA- CTTTC-3[prime]
We thank Paige Kaplan for WBS samples, Hans Lehrach for cosmid filters and clones, M.Josh Cisco and Pilar Casas for technical assistance, Stephen Scherer and Ananda Roy for prepublication manuscripts and Jonathan Eisen and Virginia Walbot for helpful discussions. The work was supported by grants from the US National Institutes of Health, the Spanish Fondo de Investigaciones de la S.S. and by the Howard Hughes Medical Institute.
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*To whom correspondence should be addressed at: Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, CA 94305-5323, USA. Tel: +1 650 725 8089; Fax: +1 650 725 8112; Email: francke@cmgm.stanford.edu
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N. PEZZI, I. PRIETO, L. KREMER, L. A. PÉREZ JURADO, C. VALERO, J. DEL MAZO, C. MARTÍNEZ-A, and J. L. BARBERO STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion
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D.-W. Kim and B. H. Cochran Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fos Promoter
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Y.-Q. WU, E. NICKERSON, L. G SHAFFER, K. KEPPLER-NOREUIL, and A. MUILENBURG A case of Williams syndrome with a large, visible cytogenetic deletion
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L. T. Reiter, T. Liehr, B. Rautenstrauss, H. M. Robertson, and J. R. Lupski Localization of mariner DNA Transposons in the Human Genome by PRINS
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K METCALFE Williams syndrome: an update on clinical and molecular aspects
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L. Edelmann, R. K. Pandita, E. Spiteri, B. Funke, R. Goldberg, N. Palanisamy, R. S. K. Chaganti, E. Magenis, R. J. Shprintzen, and B. E. Morrow A common molecular basis for rearrangement disorders on chromosome 22q11
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C. D. Novina, S. Kumar, U. Bajpai, V. Cheriyath, K. Zhang, S. Pillai, H. H. Wortis, and A. L. Roy Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase
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A Botta, G Novelli, A Mari, A Novelli, M Sabani, J Korenberg, L R Osborne, M C Digilio, A Giannotti, and B Dallapiccola Detection of an atypical 7q11.23 deletion in Williams syndrome patients which does not include the STX1A and FZD3 genes
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U. DeSilva, H. Massa, B. J. Trask, and E. D. Green Comparative Mapping of the Region of Human Chromosome 7 Deleted in Williams Syndrome
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C. D. Novina, V. Cheriyath, and A. L. Roy Regulation of TFII-I Activity by Phosphorylation
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R. Mazzarella and D. Schlessinger Pathological Consequences of Sequence Duplications in the Human Genome
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V. Cheriyath, C. D. Novina, and A. L. Roy TFII-I Regulates Vbeta Promoter Activity through an Initiator Element
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D.-W. Kim, V. Cheriyath, A. L. Roy, and B. H. Cochran TFII-I Enhances Activation of the c-fos Promoter through Interactions with Upstream Elements
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V. Cheriyath and A. L. Roy Alternatively Spliced Isoforms of TFII-I. COMPLEX FORMATION, NUCLEAR TRANSLOCATION, AND DIFFERENTIAL GENE REGULATION
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V. Cheriyath and A. L. Roy Structure-Function Analysis of TFII-I. ROLES OF THE N-TERMINAL END, BASIC REGION, AND I-REPEATS
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