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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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Activation of SNAT2 transcription by the AAR does not require enhanced recruitment of Mediator. HepG2 cells were incubated in MEM or MEM lacking histidine for 1 h, 2 h, 4 h or 8 h and then MED1 or MED23 association with the SNAT2 promoter or AARE region was monitored by ChIP analysis (a). Immunoprecipitation with a nonspecific IgG (n/s IgG) served as a negative control. MCF-7 cells were incubated in DMEM or DMEM plus 100 nM β-estradiol (E2) for 1 h and MED1, MED23 or CDK8 association with the pS2 promoter was monitored by ChIP analysis (b). On the left side, the data are presented as the ratio of protein bound to total input DNA. On the right side, the same data are shown as the fold change induced by E2 treatment. MCF-7 cells were incubated in DMEM or DMEM lacking histidine for 2 h and MED1, MED23 or CDK8 association with the SNAT2 gene was monitored by ChIP (c).
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Figure 3: Activation of SNAT2 transcription by the AAR does not require enhanced recruitment of Mediator. HepG2 cells were incubated in MEM or MEM lacking histidine for 1 h, 2 h, 4 h or 8 h and then MED1 or MED23 association with the SNAT2 promoter or AARE region was monitored by ChIP analysis (a). Immunoprecipitation with a nonspecific IgG (n/s IgG) served as a negative control. MCF-7 cells were incubated in DMEM or DMEM plus 100 nM β-estradiol (E2) for 1 h and MED1, MED23 or CDK8 association with the pS2 promoter was monitored by ChIP analysis (b). On the left side, the data are presented as the ratio of protein bound to total input DNA. On the right side, the same data are shown as the fold change induced by E2 treatment. MCF-7 cells were incubated in DMEM or DMEM lacking histidine for 2 h and MED1, MED23 or CDK8 association with the SNAT2 gene was monitored by ChIP (c).

Mentions: Mediator complex is thought to link enhancer-associated activator proteins with the promoter-bound general transcription machinery (2,18) and in yeast it has been proposed to be particularly relevant for stress-induced genes (3). Consequently, it was plausible that enhanced Mediator recruitment is required for AARE-containing genes that respond to amino acid stress in mammalian cells. To investigate Mediator association with the SNAT2 gene in HepG2 hepatoma cells, antibodies specific for the Mediator subunits MED1 or MED23 were used for ChIP analysis in cells incubated for 0–8 h in complete MEM or MEM lacking histidine (Figure 3a). At either the promoter or the AARE region of the gene, the association for either Mediator subunit was 0.01 or less (ratio to input DNA), regardless of whether the cells were amino acid deprived. This level of detection is about the same as the background value obtained by incubation with a nonspecific antibody. These results suggest that there may be little if any association of Mediator to the SNAT2 promoter or AARE enhancer regions in the basal or ‘fed’ state, and more importantly, there is no additional recruitment following amino acid stress.Figure 3.


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)

Activation of SNAT2 transcription by the AAR does not require enhanced recruitment of Mediator. HepG2 cells were incubated in MEM or MEM lacking histidine for 1 h, 2 h, 4 h or 8 h and then MED1 or MED23 association with the SNAT2 promoter or AARE region was monitored by ChIP analysis (a). Immunoprecipitation with a nonspecific IgG (n/s IgG) served as a negative control. MCF-7 cells were incubated in DMEM or DMEM plus 100 nM β-estradiol (E2) for 1 h and MED1, MED23 or CDK8 association with the pS2 promoter was monitored by ChIP analysis (b). On the left side, the data are presented as the ratio of protein bound to total input DNA. On the right side, the same data are shown as the fold change induced by E2 treatment. MCF-7 cells were incubated in DMEM or DMEM lacking histidine for 2 h and MED1, MED23 or CDK8 association with the SNAT2 gene was monitored by ChIP (c).
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Figure 3: Activation of SNAT2 transcription by the AAR does not require enhanced recruitment of Mediator. HepG2 cells were incubated in MEM or MEM lacking histidine for 1 h, 2 h, 4 h or 8 h and then MED1 or MED23 association with the SNAT2 promoter or AARE region was monitored by ChIP analysis (a). Immunoprecipitation with a nonspecific IgG (n/s IgG) served as a negative control. MCF-7 cells were incubated in DMEM or DMEM plus 100 nM β-estradiol (E2) for 1 h and MED1, MED23 or CDK8 association with the pS2 promoter was monitored by ChIP analysis (b). On the left side, the data are presented as the ratio of protein bound to total input DNA. On the right side, the same data are shown as the fold change induced by E2 treatment. MCF-7 cells were incubated in DMEM or DMEM lacking histidine for 2 h and MED1, MED23 or CDK8 association with the SNAT2 gene was monitored by ChIP (c).
Mentions: Mediator complex is thought to link enhancer-associated activator proteins with the promoter-bound general transcription machinery (2,18) and in yeast it has been proposed to be particularly relevant for stress-induced genes (3). Consequently, it was plausible that enhanced Mediator recruitment is required for AARE-containing genes that respond to amino acid stress in mammalian cells. To investigate Mediator association with the SNAT2 gene in HepG2 hepatoma cells, antibodies specific for the Mediator subunits MED1 or MED23 were used for ChIP analysis in cells incubated for 0–8 h in complete MEM or MEM lacking histidine (Figure 3a). At either the promoter or the AARE region of the gene, the association for either Mediator subunit was 0.01 or less (ratio to input DNA), regardless of whether the cells were amino acid deprived. This level of detection is about the same as the background value obtained by incubation with a nonspecific antibody. These results suggest that there may be little if any association of Mediator to the SNAT2 promoter or AARE enhancer regions in the basal or ‘fed’ state, and more importantly, there is no additional recruitment following amino acid stress.Figure 3.

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