c-Myc target gene specificity is determined by a post-DNAbinding mechanism - PubMed (original) (raw)

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

K E Boyd et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

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Figures

Figure 1

The mouse cad promoter displays E box-dependent growth-regulated transcriptional activity. (A) Sequence analysis of the mouse cad-promoter region from −112 to +129. Numbering of nucleotides is based on the putative start of transcription at +1 (indicated by the bent arrow). Transcription factor-binding sites, which are conserved between the murine and hamster cad promoters are indicated as follows: Sp1-binding sites are double underlined, the initiator element is underlined, and the E box is boxed. (B) Expression of cad mRNA displays late serum-response kinetics in mouse NIH 3T3 cells. Cytoplasmic RNA (10 μg), harvested at the indicated times after serum stimulation of quiescent NIH 3T3 cells or tRNA (10 μg) was hybridized to a murine cad riboprobe. Unprotected RNA was removed by digestion with RNase A. Protection of murine cad mRNA results in a 154-nt product, as indicated by the arrow. (C) Activated transcription from the mouse cad promoter in S phase requires the E box. Murine cad-promoter sequences from −105 to +84 or from −105 to +57 (indicated in bold in A) were fused upstream of the luciferase cDNA and transiently transfected into NIH 3T3 cells. After transfection, cells were incubated in starvation media (DMEM + 0.5% calf serum) for 48 hr, stimulated with the addition of 10% calf serum for 16 hr (corresponding to S phase of the growth cycle), and harvested for measurement of luciferase activity. The fold induction at 16 hr is reported as the ratio of luciferase activity from serum stimulated cells to the activity of the same promoter construct in serum-starved cells. Average fold induction and standard error was calculated from three independent experiments.

Figure 2

Myc binds to the cad promoter in vivo after serum stimulation of quiescent cells. Shown is the protocol for the formaldehyde crosslinking and chromatin immunoprecipitation assay used to detect protein binding to single copy genes. Formaldehyde-crosslinked chromatin was prepared from the same number of NIH 3T3 cells that were serum starved (0 hr) or serum starved and then stimulated for 4, 8, or 12 hr. Crosslinked chromatin from each time point was incubated with antibodies to Myc, USF1, or in the absence of antibody (none). In this experiment, E2F4 antibody (highlighted by the asterisk) was only incubated with the 12-hr chromatin. Immunoprecipitates from each antibody were aliquotted and then analyzed by PCR with primers specific for the cad or cyclin B promoters. To verify that at each time point an equivalent amount of chromatin was used in the immunoprecipitations, a sample representing 0.02% of the total input chromatin (input) was included in the PCR reactions.

Figure 3

Transcription from the cad promoter is activated by the transactivation domain of Myc but not USF1. Asynchronously growing NIH 3T3 cells were transiently cotransfected with 0.5 μg of cadG4luc reporter plasmid and 2.5 μg of Gal4-Myc, Gal4-USF1, and Gal4-TFE3 expression plasmid. The cells were harvested for luciferase activity at 48 hr after transfection. For each experiment, activation of the cad reporter by the Gal4-fusion proteins was normalized to the activity of the Gal4 DNA-binding domain on the same reporter. Average activation and SEM was calculated from seven to 10 independent experiments by using multiple DNA preparations.

Figure 4

Myc binds to the human cad and dhfr promoters in vivo. Crosslinking analysis of Myc, Max, and E2F4 binding at the human cad and dhfr promoters in log phase HeLa cells. Equivalent amounts of formaldehyde-crosslinked chromatin from asynchronously growing HeLa cells was incubated with antibodies to Myc, Max, E2F4, or in the absence of antibody (none). Immunoprecipitation of the human cad and dhfr promoters by each antibody was analyzed in parallel PCR reactions by using primers specific to the cad or dhfr promoters. The input sample (input) contains 0.02% of the total input chromatin as PCR template for each set of primers.

Figure 5

Transcriptional activation by Myc is dependent on the context of Myc binding. (A) The transactivation domain of Myc cannot activate expression from the dhfr promoter from the distal upstream position. Asynchronous NIH 3T3 cells were transiently cotransfected with 2.5 μg of dhfrG4luc or −375G4dhfrluc reporter and 5.0 μg of Gal4-Myc expression plasmid. Cells were harvested for luciferase activity 48 hr after transfection. For each experiment, activation of the dhfr reporter by Gal4-Myc was normalized to the activity of the Gal4 DNA-binding domain on the same reporter. Data presented was obtained from five to seven independent experiments, using multiple DNA preparations. (B) Activation of cad expression is less sensitive to the position of Myc binding. Asynchronous NIH 3T3 cells were transiently cotransfected with 0.5 μg of cadG4luc or −335cadG4luc reporter and 2.5 μg of Gal4-Myc expression plasmid. Cells were harvested and analyzed as described above. Data presented was obtained from eight to nine independent experiments.

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c-Myc target gene specificity is determined by a post-DNAbinding mechanism - PubMed (original) (raw)