contributed equally to this work Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3-deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild-type mice. Histological examinations of individual organs in Sp3 -/-mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast-specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. Recently, the mouse Sp3 gene has been disrupted by homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced defect in late tooth formation. Here we show that Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. The transcription factor - core binding factor 1 (Cbfa1)--that is essential for bone formation, however, is expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is significantly diminished. Our studies establish Sp3 as an essential transcription factor for late bone development.

Background: Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutaminerich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues.

gene in HeLa cells on CREB Binding ProteinThe HKE4 gene maps to the centromeric side of the human M H C class I1 region, proximal to the HLA-DPB2 gene. The gene, that encodes a transmembrane protein, is transcribed from a CpG-rich region. Here, we describe the characterization of this region that contains several putative binding sites for the transcription factor Spl, as well as sequences closely resembling the consensus RARE, TRE, VDRE, AP-1 and NF-KP binding sites.The transcriptional activity of the region was analyzed in transient expression assays of HKE4 promoter-luciferase reporter constructs. The transfection experiments revealed that a single G C box is sufficient for efficient transcription of the gene both in HeLa and CV-1 cells. Deletion of this site caused considerable loss of the transcriptional activity of the constructs in both cell lines. HeLa transfected cells were then treated with either NEM, TNF-a or a combination of both and, as previously shown for its murine homologue, negative regulation of the gene by TNF-a was detected. Prevention of the TNF-a induced repression of the gene by the highly specific inhibitor of the p38 MAP Kinase SB203580 was also observed. Thyroid hormone effect on the transcriptional activity of the constructs was also tested in CV-1 cells cotransfected with a T H R expression vector.The Sp family of transcription factors is composed of four proteins (Spl, Sp2, Sp3 and Sp4) characterised by similar structural features (1).Previously the Spl and the Sp4 gene has been disrupted in vivo, resulting in surprisingly different phenotypes (2,3). To elucidate the specific functions of Sp3 in vivo, we have generated Sp3-deficient mice by targeted disruption of the Sp3 gene (4). Heterozygous Sp3+/-mice appear normal. Homozygous Sp3-/-mice are growth retarded and invariably die at birth of respiratory failure. The cause for the breathing defect remains obscure since only minor morphological alterations were observed in the lung. Histological examinations of other organs show skull and tooth defects. Most significantly, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblastspecific gene products. To investigate further the functional role of Sp3, we have generated Sp3-deficient embryonic stem (ES) cells and mouse embryonic fibroblasts (MEFs).In vitro differentiation of Sp3deficient ES cells did not exhibit any obvious morphological difference compared to wild type ES cells. Screening of cDNA arrays using RNA from wild type and Sp3-/-ES cells identified potential target genes of Sp3.Ribonucleotide reductase (RNR) is the key enzyme in the formation of all four deoxyribonucleotides required for D N A synthesis. The balanced pool of D N A precursors supplied by RNR is necessary for the normal replication and repair of DNA. RNR is an obvious target for anti-proliferative treatment.The active enzyme consists of two non-identical subunits called R1 and R2. Expression of mammalian ribonucleotide reductase is regulated in a cell cycle specific manner. The t...