In the human genome three Sp gene pairs had been described previously; being maximally separated by 3.2 Mbp, transcribed in opposite directions, being orientated 5' to 5' manner and colocalized with a specific HOX gene clusters [8
]. Sp3 and Sp5 are located on chromosome 2q31.1; Sp1 and Sp7 on 12q13.13 and Sp2-Sp6 on 17q21.3-q22. A search for a partner to Sp4 located on 7q21.3-q22 revealed a putative eighth Sp gene most similar to Sp7 suggesting that Sp8, like Sp7, may play a role also in skeletal development. Consequently, we used RT-PCR to amplify Sp8 cDNAs from a human osteosarcoma cell line and isolated two transcript variants clones that encode a full-length protein (long isoform) Sp8L and an amino-terminal truncated protein (short isoform) Sp8S, with 508 and 490 residues respectively. We found a glycine-165 deletion mutation in an additional cDNA clone isolated from a patient with osteoporosis. The length of this poly-glycine region is conserved in primates and rodents, but is absent in fish. We speculate that this Sp8 mutation, and other mutations yet to be discovered, may play a role in susceptibility to osteoporosis, and since Sp8 plays an important role in neuropore closure be a candidate gene for spina bifida [9
The sequence of our Sp8L clone is similar to that of two other human clones, but the other two clones lack 170 and 128 bp of the low-complexity, B domain (GenBank accession Nos. BAB71297 and AAH38669). These deletions may be caused by incomplete reverse transcriptase reactions occurring during cDNA library synthesis, presumably because the high GC content (81%) generates strong secondary structure in the mRNA in this region. These deletion-carrying clones are unlikely to be generated by the presence of cryptic intron(s) since no suitable donor or acceptor splice sites are present in the genomic DNA sequence. One of the deletion-carrying clones, AAH38669, possesses a polymorphism, cac>cgc, at nucleotide 1263 that results in a His>Arg mutation at residue 448. His-448 is located in the third zinc- finger and is conserved in all Sp proteins and, by homology with Sp1, is likely to contact DNA [18
]. This mutated protein would be expected to be deleterious, having reduced affinity for GC-box binding sites in promoter regions.
We found that in man Sp8 has two transcript variants utilising two different first exons. Sequence analyses of mouse and zebrafish genomic and EST data support this gene structure for Sp8 in vertebrates and not a gene structure with two untranslated 5' exons that has been suggested for murine Sp8 [10
]. Interestingly, the drosophila D-Sp1 gene, a Sp8 homologue, also has two transcript variants, encoding long and short protein isoforms (accession No. AE003448) [22
]. Both Sp8 and Sp7 have a similar exon structure, being three-exon genes with two transcript variants with different first exons [8
]. The long protein isoforms use translated first exons that encodes seven residues and the short protein isoforms use untranslated first exons; overall, the proteins have 39.5% identity and 65.9% similarity. Both the Sp8S and Sp7S proteins lack an 18-residue amino-terminus thereby disrupting a hydrophobic region termed the Sp domain that is conserved in other Sp proteins. The conservation of the two protein isoforms through evolution suggests that they have differing roles. Although the function of this hydrophobic region is unknown, other zinc-finger transcription proteins often have a conserved protein-protein interaction domain at their amino-terminus (e.g. BTB or kelch domains). This indicates that the Sp motif may also be involved in a protein-protein interaction and that the short protein isoforms do not have this protein interaction domain.
Amino-terminal spliced variants expressed from separate promoters are a feature of other important transcription factors that regulate skeletal development. An oestrogen regulated protein in osteoblasts, KLF10 is another member of the Sp/Krüppel-like factor family that has two amino-terminal variant isoforms generated in a similar way to those of Sp8. These isoforms are named the TGFβ-inducible early gene (TIEG1) and the early growth response gene-alpha [23
]. Runx2 has two major amino-terminal isoforms that exert different functions during the process of osteoblast differentiation; the Runx2-type-I isoform is widely expressed in osteoprogenitor cells and active osteoblasts, whereas the Runx2-type-II isoform is restricted to cells lining mineralised bones [26
] and BMP-2 preferentially upregulates the Runx2-type-II isoform [27
Sp8 does not initiate BMP-mediated signalling during apical ectodermal ridge formation, but may function downstream of the BMP receptor-1a in the signalling cascade [9
]. The signalling events downstream of the BMP receptor that result in tissue-specific gene expression and skeletal development have been only partially elucidated. We found that Sp8 expression was induced by 100 ng/mL BMP-2 only after 24 hours in C3H10T1/2 cells. BMP-2 has been shown to induce several other transcription factors that promote differentiation such as Runx2/Cbfa1, Sp7/osterix and ZNF450 in addition to the negative regulator Id1 [7
]. Induction of these genes in C3H10T1/2 cells occurs within 4 hours, preceding that of Sp8, suggesting that Sp8 is not directly regulated by BMP signalling and that it may be induced by one or more of the BMP-early-induced genes.
The Sp8 gene has a wider phylogenetic distribution than Sp7, being found in coelomates, whereas Sp7 is limited to vertebrates. In view of the close similarities between human Sp8 and Sp7 in gene structure and amino acid sequence we speculate that they evolved from an ancestral Sp8 gene during a duplication of a Sp/Hox gene cluster [31
]. The Sp8 gene has retained its function in regulating appendage/limb growth [9
] and that Sp7 has subsequently evolved a novel function, namely, regulating cartilage/bone formation [7