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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.

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MED15 is not required for induction of SNAT2 transcription by amino acid limitation. MCF-7 cells were treated for 24 h with either ‘control’ siRNA (siControl) or siRNA for MED15, incubated in fresh DMEM for another 24 h, and then incubated in either DMEM, DMEM lacking histidine or DMEM plus 100 nM E2 for 4 h. Transcription activity for the pS2 and SNAT2 genes was analyzed as described in the Materials and methods section (a). To determine the decline in expression for MED15, mRNA was analyzed by RT–qPCR (b). MCF-7 cells were incubated in medium with or without histidine for 8 h, as well as in control medium or medium containing 100 nM E2 for 1 h and then ChIP analysis was performed for the SNAT2 promoter, SNAT2 AARE and the pS2 promoter to determine the degree of MED15 association (c).
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Figure 6: MED15 is not required for induction of SNAT2 transcription by amino acid limitation. MCF-7 cells were treated for 24 h with either ‘control’ siRNA (siControl) or siRNA for MED15, incubated in fresh DMEM for another 24 h, and then incubated in either DMEM, DMEM lacking histidine or DMEM plus 100 nM E2 for 4 h. Transcription activity for the pS2 and SNAT2 genes was analyzed as described in the Materials and methods section (a). To determine the decline in expression for MED15, mRNA was analyzed by RT–qPCR (b). MCF-7 cells were incubated in medium with or without histidine for 8 h, as well as in control medium or medium containing 100 nM E2 for 1 h and then ChIP analysis was performed for the SNAT2 promoter, SNAT2 AARE and the pS2 promoter to determine the degree of MED15 association (c).

Mentions: Zhang et al. (22) have shown that when Sin4p, a protein that links the ‘tail’ module to the body module in yeast, is deleted from the genome, a triad of proteins that make up the remainder of the tail (gal11/Med2/Pgd1), can be recruited to and activate transcription from GCN4-induced genes independently of the rest of the Mediator complex. Although mammalian cells may not have paralogs to Med2 and Pgd1 (1,18,23), to determine if MED15, the human counterpart to yeast gal11, was recruited to SNAT2 independently of the remainder of Mediator, siRNA knockdown and ChIP analysis were employed for this subunit as well. The data show that despite a 50–80% reduction of the MED15 expression (Figure 6b), the activated transcription from the pS2 gene by E2 and transcription from the SNAT2 gene was unaffected (Figure 6a). ChIP assays for MED15 (antibody from Santa Cruz Biotechnology) association with the SNAT2 promoter or AARE region revealed a relatively low level of binding (Figure 6c), yielding values that were comparable to those for a nonspecific IgG (Figure 3), and there was no additional recruitment of MED15 following amino acid limitation. When ChIP analysis was performed on the pS2 promoter to determine if MED15 recruitment was enhanced after E2 treatment, in a manner similar to other Mediator subunits shown in Figure 3, no association of MED15 with the pS2 gene was observed (Figure 6c). To extend this result, a second MED15 antibody was tested (Sigma Chemical Company), but the results were the same (data not shown).Figure 6.


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)

MED15 is not required for induction of SNAT2 transcription by amino acid limitation. MCF-7 cells were treated for 24 h with either ‘control’ siRNA (siControl) or siRNA for MED15, incubated in fresh DMEM for another 24 h, and then incubated in either DMEM, DMEM lacking histidine or DMEM plus 100 nM E2 for 4 h. Transcription activity for the pS2 and SNAT2 genes was analyzed as described in the Materials and methods section (a). To determine the decline in expression for MED15, mRNA was analyzed by RT–qPCR (b). MCF-7 cells were incubated in medium with or without histidine for 8 h, as well as in control medium or medium containing 100 nM E2 for 1 h and then ChIP analysis was performed for the SNAT2 promoter, SNAT2 AARE and the pS2 promoter to determine the degree of MED15 association (c).
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Related In: Results  -  Collection

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Figure 6: MED15 is not required for induction of SNAT2 transcription by amino acid limitation. MCF-7 cells were treated for 24 h with either ‘control’ siRNA (siControl) or siRNA for MED15, incubated in fresh DMEM for another 24 h, and then incubated in either DMEM, DMEM lacking histidine or DMEM plus 100 nM E2 for 4 h. Transcription activity for the pS2 and SNAT2 genes was analyzed as described in the Materials and methods section (a). To determine the decline in expression for MED15, mRNA was analyzed by RT–qPCR (b). MCF-7 cells were incubated in medium with or without histidine for 8 h, as well as in control medium or medium containing 100 nM E2 for 1 h and then ChIP analysis was performed for the SNAT2 promoter, SNAT2 AARE and the pS2 promoter to determine the degree of MED15 association (c).
Mentions: Zhang et al. (22) have shown that when Sin4p, a protein that links the ‘tail’ module to the body module in yeast, is deleted from the genome, a triad of proteins that make up the remainder of the tail (gal11/Med2/Pgd1), can be recruited to and activate transcription from GCN4-induced genes independently of the rest of the Mediator complex. Although mammalian cells may not have paralogs to Med2 and Pgd1 (1,18,23), to determine if MED15, the human counterpart to yeast gal11, was recruited to SNAT2 independently of the remainder of Mediator, siRNA knockdown and ChIP analysis were employed for this subunit as well. The data show that despite a 50–80% reduction of the MED15 expression (Figure 6b), the activated transcription from the pS2 gene by E2 and transcription from the SNAT2 gene was unaffected (Figure 6a). ChIP assays for MED15 (antibody from Santa Cruz Biotechnology) association with the SNAT2 promoter or AARE region revealed a relatively low level of binding (Figure 6c), yielding values that were comparable to those for a nonspecific IgG (Figure 3), and there was no additional recruitment of MED15 following amino acid limitation. When ChIP analysis was performed on the pS2 promoter to determine if MED15 recruitment was enhanced after E2 treatment, in a manner similar to other Mediator subunits shown in Figure 3, no association of MED15 with the pS2 gene was observed (Figure 6c). To extend this result, a second MED15 antibody was tested (Sigma Chemical Company), but the results were the same (data not shown).Figure 6.

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