Expression and developmental requirement for transcription factor Sp2

Abstract

The Sp-family of DNA-binding proteins is comprised by nine members, and controls the expression of many mammalian genes. Most Sp-family members have been well studied with respect to their patterns of expression as well as requirement for mammalian development. However, little information regarding the expression and function of Sp2 was available. Our laboratory has reported that Sp2 is widely expressed in murine and human cell lines, Sp2 DNA-binding activity and trans-activation are negatively regulated in vitro, and that the vast majority of Sp2 localizes to sub-nuclear foci associated with the nuclear matrix. To extend these studies, expression of the mouse Sp2 locus was analyzed in detail and the requirement for Sp2 for mouse development was evaluated via the creation of a conditional "knock-out" mouse strain. To initiate the analysis of Sp2 I first identified transcriptional start sites using a PCR-assisted (5'RACE) strategy. Sequencing of 5’RACE clones showed that transcriptional start sites clustered within three regions of the Sp2 locus producing three types of transcripts: Exon 2A (type I), Exon 4 (type II) and Exon 5 (type III). Synthesis of type I transcripts was shown to be directed by a promoter that contains sequences encoding at least four discrete enhancer and inhibitory elements. Additional promoters were not identified within the Sp2 locus. Using RT-PCR and RNA in situ hybridization assays, Sp2 was shown to be widely expressed at embryonic and post-natal stages and it's expression was noted to be particularly concentrated in brain sub-regions. Sp2 conditional "knock-out" mice were generated via the insertion of loxP sites flanking the first two Sp2 coding exons. The developmental consequences of loss of Sp2 function were evaluated following matings with three Cre recombinase-carrying strains that express Cre in a widespread (CMV-Cre) or tissue-restricted fashion (Keratin 14-Cre and Keratin14-Cre/Estrogen receptor fusion). Sp2 hemizygous animals are indistinguishable from wild-type animals, whereas nullizygous animals perish early in gestation. These data indicate that Sp2 is an essential gene at the organismal level, yet we have also shown that Sp2 nullizygous cells are viable in adult, mosaic animals. Constitutive (Keratin 14-Cre) or induced (Keratin 14-Cre/Esr) expression of the Cre recombinase in basal keratinocytes resulted in a dramatic increase in stem cell proliferation and the loss of Keratin 5 and Keratin 15 expression in these cells. Taken together, we conclude that Sp2 is required for early embryogenesis and regulates the proliferation and differentiation of adult progenitor cells.