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P J Farnham

Publications and source records attributed to P J Farnham.

At least 37 records · Page 2Linked to original sources
PubMed1997

E2F-mediated growth regulation requires transcription factor cooperation.

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.

3T3 Cells↗
PubMed1997

Position-dependent transcriptional regulation of the murine dihydrofolate reductase promoter by the E2F transactivation domain.

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site.

3T3 Cells↗
PubMed1997

Myc versus USF: discrimination at the cad gene is determined by core promoter elements.

Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which is encoded by the cad gene, is required for the first three rate-limiting steps of de novo pyrimidine biosynthesis. It has been previously demonstrated that cad transcription increases at the G1/S-phase boundary, as quiescent cells reenter the proliferative cell cycle. The growth-responsive element has been mapped to an E box at +65 in the hamster cad promoter. Using an in vivo UV cross-linking and immunoprecipitation assay, we show that Myc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine whether binding of Myc-Max or USF is critical for cad growth regulation, we analyzed promoter constructs which contain mutations in the nucleotides flanking the E box. We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vitro correlates with a loss of cad G1/S-phase transcriptional activation. This result supports the conclusion that binding of Myc-Max, but not USF, is essential for cad regulation. Our investigations demonstrate that the endogenous cad E box can be bound by more than one transcription factor, but growth-induced cad expression is achieved only by Myc.

3T3 Cells↗
PubMed1996

Transcriptional regulation of the dihydrofolate reductase gene.

As cells approach S phase, many changes occur to create an environment conducive for DNA synthesis and commitment to cell division. The transcription rate of many genes encoding enzymes involved in DNA synthesis, including the dihydrofolate reductase (dhfr) gene, increases at the G1/S boundary of the cell cycle. Although a number of transcription factors interact to finely tune the levels of dhfr RNA produced, two families of transcription factors, Sp1 and E2F, play central roles in modulating dhfr levels. A region containing several Sp1-binding sites is required for both regulated and basal transcription levels. In contrast, the E2F-binding sites near the transcription start site are required only for regulated transcription. A model is presented for the regulation of the dhfr gene which may also pertain to other cell cycle-associated genes.

Animals↗
PubMed1996

Introduction to the E2F family: protein structure and gene regulation.

E2F is a heterodimer composed of two partners, such as E2F1 and DP1. Although E2F1 can bind DNA as a homodimer and increase promoter activity, optimal DNA-binding and transcriptional activity occurs in the heterodimeric form. A model (Fig. 3) for the involvement of E2F activity in cell growth control that incorporates viral oncoproteins, positive regulators of cell growth (cyclins) and negative regulators of cell growth (tumor suppressor proteins) can now be advanced. Each aspect of this model is addressed in subsequent chapters of this book. It is likely that binding of growth-suppressing proteins, such as Rb, can inhibit the transactivation potential of E2F1, either by blocking the interaction of E2F1 with a separate component of the transcription complex or by bringing a repressor domain to the transcription complex (Flemington et al. 1993; Helin et al. 1993; Weintraub et al. 1992; Zamanian and La Thangue 1993; Zhu et al. 1993). Phosphorylation or sequestration of Rb by viral oncoproteins can free E2F. The influence of viral oncoproteins on E2F activity and the regulation of the different E2F complexes is the focus of the contributions by Cobrinik and by Cress and Nevens. The interaction of the free E2F induces a bend in the DNA that may also play a role in transactivation, perhaps by bringing proteins (such as an Sp1 or CCAAT family member) separated by distance on the promoter DNA into contact (Huber et al. 1994). Because E2F target genes encode proteins critical for cell growth, deregulation of E2F activity can have severe consequences, such as apoptosis or uncontrolled proliferation. The effect of altered expression of E2F activity on the cell cycle and on tumorigenicity is the focus of the contribution by Adams and Kaelin. Finally, a comparison of E2F to the genetically well-characterized factors that regulate G1/S phase transcription in yeast is the subject of the chapter by Breeden. This volume concludes with Farnham's summary of the rapid gains in knowledge concerning the E2F gene family that have been made in the past several years and provides a series of questions and lines of investigation that will be the focus of future studies.

Animals↗
PubMed1995

Identification of cis-acting elements that can obviate a requirement for the C-terminal domain of RNA polymerase II.

We have used an in vitro RNA polymerase II (RNAP II) inhibition-restimulation assay to investigate the inability of a form of RNAP II (RNAP IIB) that lacks the conserved, C-terminal heptapeptide repeat domain (CTD) to transcribe the dihydrofolate reductase (dhfr) promoter. Our previous studies demonstrated promoter-specific responses to RNAP IIB in the inhibition-restimulation assay and suggested the existence of cis-acting elements that alleviate the requirement for the CTD. We have now identified elements from two different classes of promoters that can convert dhfr to a CTD-independent promoter. First, addition of a consensus TATA box to the dhfr promoter resulted in a promoter capable of CTD-independent transcription and increased the promoter's affinity for the general transcription factor TFIID. These results suggest that high affinity for TFIID correlates with an ability to be transcribed by RNAP IIB, supporting a proposed interaction between the CTD and TFIID. Second, transfer of a combination of two elements (located at -25 and +1) from the rep-3b promoter, which does not contain a consensus TATA box but can nonetheless be transcribed by RNAP IIB, into the dhfr promoter also allowed CTD-independent transcription. These elements do not constitute a high affinity binding site for TFIID, indicating that an additional mechanism exists to allow CTD-independent transcription. Thus, elements from two classes of CTD-independent promoters that can obviate a requirement for the CTD appear to function via distinct mechanisms. Our finding that a change in a basal element can affect a requirement for the CTD is consistent with a role for the CTD during the formation of the transcriptional preinitiation complex.

Base Sequence↗
PubMed1995

Strain-dependent differences in DNA synthesis and gene expression in the regenerating livers of CB57BL/6J and C3H/HeJ mice.

C3H/HeJ (C3H) mice are approximately 50-fold more susceptible to liver-tumor induction than C57BL/6J (B6) mice. This difference is susceptibility is a consequence of allelic differences in hepatocarcinogen sensitivity (Hcs) genes that control the growth of preneoplastic lesions in the liver. We have shown previously that these two strains differ in their responses to partial hepatectomy, which acts as a promoter of hepatocarcinogenesis in B6 mice but not in C3H mice. To determine whether there are also strain-specific differences in normal regulation of hepatic growth, we compared liver regeneration in C3H and B6 mice at the levels of DNA synthesis and gene expression. Partial hepatectomy induced a cascade of controlled events resulting in the regeneration of the liver to its original mass 11 d after surgery. We observed a two-fold greater level of DNA synthesis in C3H mice relative to B6 mice during the first peak of DNA synthesis, which occurred 35 h after hepatectomy in both strains. While the c-fos transcript was readily induced in both strains, there was a reduction in the expression of the late response genes E2F1 and dihydrofolate reductase in the livers of B6 mice when compared with the expression of these transcripts in the livers of C3H mice. The differential regulation of E2F1 between B6 and C3H mice may indicate that the Hcs genes and E2F1 function in the same signal transduction pathway of normal growth control.

Animals↗
PubMed1995

An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants.

To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G1/S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at +55 to +75 (+55/+75) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed down-stream of the transcription start site. The same oligonucleotide conferred less G1/S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad +55/+75 element in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G1/S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G1/S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.

3T3 Cells↗
PubMed1995

The bidirectionally transcribed dihydrofolate reductase and rep-3a promoters are growth regulated by distinct mechanisms.

The mouse dihydrofolate reductase (dhfr) gene possesses a bidirectional promoter that produces functional transcripts in the opposite direction. These opposite strand transcripts encode the Rep-3 gene product, a protein that has homology to DNA mismatch repair enzymes. The core of the bidirectional promoter consists of four consensus binding sites for the transcription factor Sp1. These binding sites have been shown to be important for basal transcription from both the rep-3a and dhfr promoters. Extensive characterization of the dhfr promoter has shown that growth-dependent regulation requires the E2F binding sites that flank the transcription initiation site. Here we show that endogenous rep-3a mRNA and the rep-3a promoter are growth regulated, in a manner very similar to the regulation of the dhfr mRNA and promoter region. However, we find that the E2F sites required for dhfr regulation are dispensible for regulation of the rep-3a promoter. Instead, we have shown that the rep-3a initiation region is critical for the G1S phase-specific activation of this promoter. Gel mobility shift experiments indicate that a member of the Sp1 family of transcription factors binds to the rep-3a initiation region, suggesting that this family of transcription factors may play a role in cell growth control.

3T3 Cells↗
PubMed1995

v-Raf activates transcription of growth-responsive promoters via GC-rich sequences that bind the transcription factor Sp1.

The serine/threonine kinase, Raf-1, is a component of intracellular signaling pathways that control responses to extracellular stimuli. Previously, we have shown that serum-induced transcription from the murine rep-3b and human mdr1 promoters is Raf-dependent and that the activated Raf kinase, v-Raf, induces transcription of mdr1 via a GC-rich element. We now demonstrate that GC-rich sequences in the rep-3b promoter are both necessary and sufficient for induction by v-Raf. The GC-rich, v-Raf-responsive elements of rep-3b and mdr1 bind the general transcription factor Sp1 in electromobility shift assays. Mutation of a minimal GC-rich element abolished inducibility by v-Raf and eliminated binding by the transcription factor Sp1. However, Sp1 binding activity following serum stimulation of quiescent NIH 3T3 cells was unchanged, suggesting that mitogenic signals may stimulate the transactivation potential of prebound Sp1.

3T3 Cells↗
PubMed1994

Inappropriate transcription from the 5' end of the murine dihydrofolate reductase gene masks transcriptional regulation.

Using the nuclear run-on assay we found that in proliferating cells the transcription rate in the 5' end of the murine dihydrofolate reductase (dhfr) gene was approximately ten-fold higher than in the 3' end of the gene, suggesting transcriptional attenuation within the dhfr gene. However, when the transcription rate was measured by pulse-labeling, the rate was uniform throughout the gene, and the 5' dhfr signal was approximately ten-fold lower relative to a control gene signal than in the run-on assay. Previously, the activity of a dhfr promoter linked to a luciferase reporter gene was shown to increase about ten-fold at the G1/S-phase boundary following stimulation of serum-starved cells. To determine if the run-on procedure would detect growth regulation of the endogenous dhfr gene, serum-starved and -stimulated NIH 3T3 cells were analyzed. Using a dhfr 5' end probe no difference in transcription rate between these growth states was detected and the dhfr 3' end probe did not detect signal above background. In a cell line that was amplified at the dhfr locus, the transcription rate in the 5' end of the gene increased less than two-fold in stimulated cells, but the rate in the 3' end of the gene increased five- to seven-fold. Therefore, the dhfr gene is growth regulated at the level of transcription, but the nuclear run-on assay was only able to detect a difference in transcription rate in the 3' end of the gene in amplified cells. We suggest that isolation of nuclei may activate dhfr transcription complexes that normally are activated only at the G1/S-phase boundary.

3T3 Cells↗
PubMed1994

Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter.

To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases approximately 80-fold at the G1/S-phase boundary and that this activation is, in part, regulated by G0-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.

3T3 Cells↗
PubMed1994

Start site selection at the TATA-less carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase promoter.

Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.

3T3 Cells↗
PubMed1994

Cloning, chromosomal location, and characterization of mouse E2F1.

E2F has been implicated in growth control because of its association with the retinoblastoma protein and the presence of E2F binding sites in the promoters of several growth-regulated genes. Proteins that bind to an E2F site have been cloned from human and mouse cells. However, these two proteins (human E2F1 and mouse DP-1) are quite different in sequence. We have now cloned a mouse cDNA encoding a protein 86% identical to the human E2F1 protein. The mouse E2F1 cDNA encodes a 430-amino-acid protein with a predicted molecular weight of 46,322 and detects mRNAs of 2.7 and 2.2 kb. Using primers complementary to sequences in the mouse E2F1 3' untranslated region, we mapped the mouse E2F1 gene to chromosome 2, near the Agouti and c-src loci. To understand the role of the different E2F family members in the growth of mouse NIH 3T3 cells, we examined the levels of E2F1 and DP-1 mRNAs in different stages of the cell cycle. Since the levels of E2F1 but not DP-1 mRNA correlated with changes in transcription from the dhfr promoter, we examined whether E2F1 could activate various growth-regulated promoters. We found that E2F1 could activate some (dhfr, thymidine kinase, and DNA polymerase alpha) but not all (thymidylate synthase, cad, and c-myc) of these promoters. On the basis of changes in levels of E2F1 and its ability to transactivate growth-regulated promoters, we propose that E2F1 may mediate growth factor-initiated signal transduction.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of the dihydrofolate reductase/rep-3 locus.

This review summarizes many studies that have used the mouse, hamster, or human dihydrofolate reductase locus as a model system for the study of basal or regulated transcription. This locus encodes two genes, dhfr and rep-3, that are oriented in opposite directions. The dhfr promoter and the two rep-3 promoters are highly GC-rich and do not contain consensus TATA boxes. The cis-acting elements important in basal transcription of these non-TATA box promoters have been mapped, revealing that Sp1 plays a dominant role, and that sequence elements both upstream and downstream of the initiation site contribute to transcriptional activity. Future studies will be directed toward understanding the assembly of transcription complexes on these promoters. The expression of the dhfr gene following treatment of cells with many different stimuli has been analyzed, and in many, but not all, cases the response has been at the transcriptional level. In those systems where the regulatory cis-acting element has been mapped, the E2F sites flanking the transcription initiation site play a central role in mediating the response. Future studies will be directed at characterizing the regulatory protein E2F and understanding how it interacts with the basal transcription complex at the dhfr promoter. The rep-3 gene contains two promoters that are both growth responsive: one promoter is regulated by E2F, the regulator of the other promoter has not been identified. In summary, the in-depth characterization of the 1 kb containing the dhfr and rep-3 promoters has begun to provide a detailed understanding of growth-responsive transcription.

3T3 Cells↗
PubMed1993

An inhibitory Raf-1 mutant suppresses expression of a subset of v-raf-activated genes.

The proto-oncogene Raf-1 is a cytoplasmic serine/threonine kinase implicated in the signaling process in cell proliferation. To determine if Raf-1 is sufficient and necessary to transmit mitogenic signals to growth-responsive genes, we examined the effect of constitutively activated (v-raf) or inhibitory (Raf-C4) Raf-1 proteins on reporter gene activation in transient expression assays of NIH 3T3 cells. In serum-starved cells, v-raf strongly activated transcription from the promoters of the immediate-early genes c-fos and egr-2, as well as the proximal or B promoter of the late growth response gene rep-3 (rep-3b). Two other late response gene promoters, cad and dhfr, were only modestly activated by v-raf, however. An individual serum response element from the c-fos or egr-2 promoter conferred both serum-inducibility and v-raf-responsiveness to a heterologous promoter. Consistent with the degree to which antisense c-raf-1 RNA and dominant-negative Raf-1 proteins interfere with NIH 3T3 cell proliferation, Raf-C4 reduced serum-induced transcription from the egr-2 and rep-3b promoters in a dose-dependent manner by 50%. In contrast, Raf-C4 did not significantly reduce transcription from the c-fos or cad promoters or the serum response element-driven heterologous promoters. We conclude that Raf-1 is both sufficient and necessary to activate a subset of early and late growth response genes.

3T3 Cells↗