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The number of mRNAs and proteins that can be produced from a single gene is known to be increased by the number of start sites and by multiple splicing of products. A few genes have been found to generate extraordinarily large numbers of splicing isoforms. In the human, the largest number, nearly 2000 mRNA isoforms, has been reported for the neurexin 3α gene. However, the biological significance of alternative splicing often remains unclear because many alternative transcripts contain early translational stops and are thought to be rapidly degraded. We demonstrate here that human basonuclin 2 (bn2; approved gene symbol BNC2) transcripts are initiated from six promoters, are alternatively spliced at multiple positions, and are polyadenylated at four sites. Characterization of nearly 100 bn2 mRNA isoforms suggests that each promoter, splice site, and poly(A) addition site is used independently. The bn2 gene has therefore the potential to generate up to 90,000 mRNA isoforms encoding more than 2000 different proteins. Because alternative exons affect the position of the first methionine codon, the length of the coding region, and the position of the translational stop, the encoded proteins range in size from 43 to 1211 amino acids and some bear no sequence similarity to others. PCR analysis and transient expression in HeLa cells show that the major bn2 mRNA isoforms are stable and are translated into equally stable proteins, even when the mRNA bears an early translational stop.

BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2−/− mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2−/− mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2−/− mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2−/− mice. The function of the basonuclins in the secondary hair germ is of particular interest.

Basonuclins 1 and 2 are nuclear proteins specific to vertebrates. They have recently been shown to be the orthologs of the DISCO proteins of insects. All of these proteins have an essential function in embryonic development. It is likely that they are transcription factors with multiple gene targets, some of which are transcribed by DNA polymerase I, and others by polymerase II. It remains to be found which targets are common to the two basonuclins and even to the DISCO proteins, and why certain cell types possess either basonuclin 1 or basonuclin 2, while others possess both. The implication of the basonuclins in several human diseases adds to their interest as regulatory proteins.

Basonuclin 1 and the recently discovered basonuclin 2 are vertebrate proteins with multiple paired C(2)H(2) zinc fingers. It has long been known that the zinc fingers of basonuclin 1 closely resembled those of the Drosophila disconnected and discorelated proteins, two proteins essential for head development, but the relation between the basonuclins and the disco proteins has remained unclear because the putative function of basonuclin 1 in the control of keratinocyte growth potential appeared unrelated to that of disco and there was no resemblance between basonuclin 1 and Drosophila disco outside of the zinc fingers. The recent generation of a basonuclin-2 knockout has demonstrated that basonuclin 2 shares with disco a function in head development and the availability of new arthropod genome sequences has shown that the basonuclins are the vertebrate orthologs of the insect disco proteins. All these proteins are thought to be transcription factors, and it will have to be determined to what extent they share similar targets.

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non- Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2.

Basonuclin is a zinc finger protein specific to basal keratinocytes and germ cells. In keratinocytes, basonuclin behaves as a stem cell marker and is thought to be a transcription factor that maintains proliferative capacity and prevents terminal differentiation. The human gene is located on chromosome 15. We have discovered in the chicken the existence of basonuclin 2, a basonuclin homolog. We also report the entire sequence of mouse and human basonuclin 2; the corresponding genes are located on mouse chromosome 4 and human chromosome 9. Although the amino acid sequence of basonuclin 2 differs extensively from that of basonuclin 1, the two proteins share essential features. Both contain three paired zinc fingers, a nuclear localization signal, and a serine stripe. The basonuclin 2 mRNA has a wider tissue distribution than the basonuclin 1 mRNA: it is particularly abundant in testis, kidney, uterus, and intestine. The extreme conservation of the basonuclin 2 amino acid sequence across vertebrates suggests that basonuclin 2 serves an important function, presumably as a regulatory protein of DNA transcription.