Jane Clifford Azizkhan

Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.

Induction of NF-kappa B DNA-binding activity during the G0-to-G1 transition in mouse fibroblasts.

A DNA-binding factor with properties of NF-kappa B and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 cells are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent cells with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-kappa B-like activity is not detected in nuclear extracts of proliferating cells, and thus its expression appears to be limited to the G0-to-G1 transition in 3T3 cells. These DNA-binding activities bind many of the expected NF-kappa B target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as well as a recently identified NF-kappa B binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-kappa B binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-kappa B-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the G0-to-G1 transition.

Distinct roles for Sp1 and E2F sites in the growth/cell cycle regulation of the DHFR promoter

Dihydrofolate reductase activity is required for many biosynthetic pathways including nucleotide synthesis. Its expression is therefore central to cellular growth, and it has become a key target for cancer chemotherapy. Transcription of the dihydrofolate reductase gene is regulated with growth, being expressed maximally in late G1/early S phase following serum stimulation of quiescent cells. This regulation is directed by a promoter which contains binding sites for only the transcription factors Sp1 and E2F. In this study, the role of these promoter elements in growth/cell cycle regulation of dihydrofolate transcription was addressed directly by transient transfection of Balb/c 3T3 cells with mutant promoter‐reporter gene constructs. The E2F sites were found to repress transcription in G0 and early G1 but did not contribute to the level of transcription in late G1/S phase. In contrast, Sp1 sites were able to mediate induction of transcription from the dihydrofolate reductase promoter, as well as a heterologous promoter, following serum stimulation of quiescent cells. These findings add dihydrofolate reductase to a growing list of genes at which E2F sites are primarily repressive elements and delineate a role for Sp1 sites in the growth/cell cycle regulation of transcription. J. Cell. Biochem. 67: 24–31, 1997.

Transcription factor AP2 is required for expression of the rat transforming growth factor-α gene

DNase I footprinting of the rat TGFα promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from −222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGFα promoter-reporter gene (TGFαLUC) from 50 – 90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5′-GCCNNNGGC-3′) as its center, we investigated the possibility that AP2 binding is important for TGFα promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGFαLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGFα promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.

Somatomedin-C/insulinlike growth factor I (Sm-C/IGF-I) and insulinlike growth factor II (IGF-II) mRNAs during lung development in the rat.

Somatomedins/insulinlike growth factors (Sm/IGFs) are peptide growth factors that are synthesized in multiple tissues and have been implicated in organ growth. Poly(A +)RNAs from lungs of fetal and postnatal rats were examined by dot and Northern blot analyses to investigate the local synthesis of IGFs during lung organogenesis. Blots were successively hybridized with 32P-labeled human IGF-II and mouse Sm-C/IGF-I cDNAs followed by human ubiquitin cDNAs (a control). Multiple IGF-II mRNA species of 4.7, 3.9, 3.1, 2.2, 1.75, and 1.2 kb were observed. Prenatally, the 3.9 kb species was predominant. In adult lung, the 4.7 kb species was the major species, and the 1.2 kb species was not detectable. Multiple Sm-C/IGF-I mRNA species of estimated sizes 7.5, 4.7, 1.7, and 1.0 kb were observed and their relative abundance did not change discernibly throughout development. Densitometric quantification revealed that IGF-II mRNAs were 7-20 fold more abundant in fetal lung than adult lung. In addition, they were 5-8 times more abundant than Sm-C/IGF-I mRNAs in embryonic lung, and were slightly less abundant than Sm-C/IGF-I mRNAs in adult lung. These data suggest synthesis of both Sm-C/IGF-I and IGF-II throughout lung organogenesis. Developmental changes in IGF-II mRNA abundance during lung organogenesis imply regulation at the level of gene transcription and/or mRNA stability.

SOMATOMEDIN-C|[sol]|INSULIN-LIKE GROWTH FACTORS I (Sm-C|[sol]|IGF-I) AND IGF-II mRNAs DURING LUNG DEVELOPMENT IN THE RAT

Sm-C/IGF-I and IGF-II are peptide mitogens that are synthesized in multiple organs and are implicated in regulation of organ growth. To investigate the ontogeny of their synthesis during lung development, Northern and dot blot hybridizations were performed on poly(A+)RNAs from lungs of fetal rats at 16, 17, 19, and 21 days gestation and postnatally at 0, 1, 2, 3-4, 6-7, 10, 14, 21, 28 and >50 days. A 32P-labelled rat Sm-C/IGF-I genomic fragment, a mouse Sm-C/IGF-I cDNA, and a human IGF-II cDNA were used as probes. These analyses revealed that the abundance of total Sm-C/IGF-I mRNA is 1.5 to 2.5-fold higher in lungs of fetal than postnatal animals and that multiple Sm-C/IGF-I mRNA species of estimated sizes 7.5, 4.7, 1.7 and 1.2 kb are observed at every age examined. For IGF-II, a high abundance of total hybridizing mRNA is found in fetal lung; it rapidly declines in the first week after birth to a plateau that is 20-fold less than in the fetus. Multiple lung IGF-II mRNAs of 4.7, 3.9, 2.2, 1.75 and 1.2 kb are observed. In the fetal and immediate postnatal periods, the 4.7 and 3.9 kb mRNAs are predominant. By one week postnatally, the 2.2kb mRNA is increased in relative abundance and the 1.2 kb mRNA is not detected. These data suggest synthesis of Sm-C/IGF-I and IGF-II throughout lung organogenesis. The abundance of mRNA for each peptide is developmentally regulated, and for IGF-II the developmental stage may determine the mRNA species expressed.