Limits...
Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex.

Thiaville MM, Dudenhausen EE, Awad KS, Gjymishka A, Zhong C, Kilberg MS - Nucleic Acids Res. (2008)

Bottom Line: Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription.The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment.These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT
It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

Show MeSH
Reduction in Mediator expression does not block induction of SNAT2 transcription. MCF-7 cells were treated with siRNA and incubated in DMEM, DMEM plus E2 or DMEM lacking histidine as described in Figure 4 and then pS2 (a) or SNAT2 (b) transcription activity was analyzed by RT–qPCR. Asterisks denote transcription activity in the siMed condition that is significantly different (P < 0.05) from the siControl condition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553592&req=5

Figure 5: Reduction in Mediator expression does not block induction of SNAT2 transcription. MCF-7 cells were treated with siRNA and incubated in DMEM, DMEM plus E2 or DMEM lacking histidine as described in Figure 4 and then pS2 (a) or SNAT2 (b) transcription activity was analyzed by RT–qPCR. Asterisks denote transcription activity in the siMed condition that is significantly different (P < 0.05) from the siControl condition.

Mentions: Given the limited number of Mediator antibodies that function for ChIP analysis and to obtain data from an independent approach, MCF-7 cells were treated with an siRNA specific for one of seven different Mediator subunits, MED1, MED6, MED7, MED14, MED17, MED23 or MED31. To monitor the effectiveness of the siRNA action, 48 h after transfection, cellular RNA was isolated and subjected to qRT–PCR to monitor steady state mRNA levels (Figure 4). The results demonstrate that each of the siRNA treatments was effective in reducing the corresponding Mediator subunit expression by 50–90%, relative to the siControl. For MED1 and MED6, the mRNA results were confirmed by immunoblotting for protein (data not shown). To monitor the effect of Mediator knockdown on transcription activity, cells were treated with either E2 to activate transcription from the pS2 gene (Figure 5a) or medium lacking histidine to induce SNAT2 transcription (Figure 5b). Using the pS2 gene as a positive control, the E2 transcriptional activation was blocked by >50% when MED1, MED6, MED7, MED17 and MED31 were reduced in their expression. For unknown reasons, knockdown of MED14 and MED23 had no effect on the E2 induction. In contrast, for each of the seven Mediator subunits there was no effect on the transcription activity from the SNAT2 gene in the basal or ‘fed’ state nor was the activation of the SNAT2 gene by amino acid limitation reduced (Figure 5b).Figure 4.


Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex.

Thiaville MM, Dudenhausen EE, Awad KS, Gjymishka A, Zhong C, Kilberg MS - Nucleic Acids Res. (2008)

Reduction in Mediator expression does not block induction of SNAT2 transcription. MCF-7 cells were treated with siRNA and incubated in DMEM, DMEM plus E2 or DMEM lacking histidine as described in Figure 4 and then pS2 (a) or SNAT2 (b) transcription activity was analyzed by RT–qPCR. Asterisks denote transcription activity in the siMed condition that is significantly different (P < 0.05) from the siControl condition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553592&req=5

Figure 5: Reduction in Mediator expression does not block induction of SNAT2 transcription. MCF-7 cells were treated with siRNA and incubated in DMEM, DMEM plus E2 or DMEM lacking histidine as described in Figure 4 and then pS2 (a) or SNAT2 (b) transcription activity was analyzed by RT–qPCR. Asterisks denote transcription activity in the siMed condition that is significantly different (P < 0.05) from the siControl condition.
Mentions: Given the limited number of Mediator antibodies that function for ChIP analysis and to obtain data from an independent approach, MCF-7 cells were treated with an siRNA specific for one of seven different Mediator subunits, MED1, MED6, MED7, MED14, MED17, MED23 or MED31. To monitor the effectiveness of the siRNA action, 48 h after transfection, cellular RNA was isolated and subjected to qRT–PCR to monitor steady state mRNA levels (Figure 4). The results demonstrate that each of the siRNA treatments was effective in reducing the corresponding Mediator subunit expression by 50–90%, relative to the siControl. For MED1 and MED6, the mRNA results were confirmed by immunoblotting for protein (data not shown). To monitor the effect of Mediator knockdown on transcription activity, cells were treated with either E2 to activate transcription from the pS2 gene (Figure 5a) or medium lacking histidine to induce SNAT2 transcription (Figure 5b). Using the pS2 gene as a positive control, the E2 transcriptional activation was blocked by >50% when MED1, MED6, MED7, MED17 and MED31 were reduced in their expression. For unknown reasons, knockdown of MED14 and MED23 had no effect on the E2 induction. In contrast, for each of the seven Mediator subunits there was no effect on the transcription activity from the SNAT2 gene in the basal or ‘fed’ state nor was the activation of the SNAT2 gene by amino acid limitation reduced (Figure 5b).Figure 4.

Bottom Line: Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription.The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment.These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT
It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

Show MeSH