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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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Amino acid limitation induces region-specific changes in histone acetylation, SNAT2 transcription activity and GTF binding at the SNAT2 gene. (a) The SNAT2 transcription activity and the binding of RNA Pol II, both published elsewhere (14), are provided as a graphical representation to provide a comparison with the ChIP data for the remaining GTFs. To obtain the data of (a), HepG2 cells were incubated in MEM (open circle) or MEM lacking histidine (closed circle) for 1 h, 2 h, 4 h or 8 h and protein association with the SNAT2 promoter was monitored by ChIP assay. For each of the GTF proteins shown in (a) binding to the SNAT2 AARE was also tested, but was not significantly different from the background value obtained with a nonspecific antibody. An example is illustrated by the data for TFIIB (b). Nuclear extracts were prepared and subjected to immunoblot analysis for several GTFs, those for TAFI, Pol II and TFIIB are shown as examples (c).
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Figure 1: Amino acid limitation induces region-specific changes in histone acetylation, SNAT2 transcription activity and GTF binding at the SNAT2 gene. (a) The SNAT2 transcription activity and the binding of RNA Pol II, both published elsewhere (14), are provided as a graphical representation to provide a comparison with the ChIP data for the remaining GTFs. To obtain the data of (a), HepG2 cells were incubated in MEM (open circle) or MEM lacking histidine (closed circle) for 1 h, 2 h, 4 h or 8 h and protein association with the SNAT2 promoter was monitored by ChIP assay. For each of the GTF proteins shown in (a) binding to the SNAT2 AARE was also tested, but was not significantly different from the background value obtained with a nonspecific antibody. An example is illustrated by the data for TFIIB (b). Nuclear extracts were prepared and subjected to immunoblot analysis for several GTFs, those for TAFI, Pol II and TFIIB are shown as examples (c).

Mentions: The SNAT2 gene has an AARE in intron 1 (nucleotides +709 to +717) that functions as an ATF4-responsive enhancer to activate transcription within 1 h after amino acid limitation and the transcription remains elevated for about 10–12 h (13,14). The time course of SNAT2 transcription activity and recruitment of RNA Pol II have been published previously (14), but for the purpose of comparison a graphical representation is shown in the first panel of Figure 1a. It has been suggested that in the absence of Mediator, the TFIID complex may provide the link between enhancer elements and RNA Pol II (4). For SNAT2, the activator ATF4 binds to the intronic AARE, but does not appear to interact directly with the promoter region (12). To provide mechanistic insight into assembly of the preinitiation complex following amino acid limitation, binding of the TFIIA, TFIIB, TFIID (TAF1 and TAF9) and TFIIE was measured by ChIP analysis (Figure 1a). Following amino acid removal, all of the GTFs exhibited increased binding to the SNAT2 promoter in a manner that qualitatively paralleled the enhancement in transcription activity. In contrast, as shown by the example of TFIIB (Figure 1b), none of the GTFs showed significant binding to the intronic region containing the SNAT2 AARE (TFIIA, TFIID and TFIIE, not shown). The stress-induced recruitment of the GTFs was not a consequence of a change in their absolute concentration, as illustrated by immunoblotting of nuclear extracts (Figure 1c). Increased acetylation of histone H3 (AcH3) is a general marker for induced gene expression. To monitor the localization of amino acid-dependent chromatin modification for the SNAT2 gene, ChIP analysis for AcH3 was performed at specific regions across the SNAT2 gene 8 h after amino acid limitation (Figure 2a). The SNAT2 promoter exhibited the largest increase in AcH3, but acetylation was also elevated at the AARE. In contrast, at regions only about 1 kb upstream or downstream within the gene, there was little or no increase in histone modification relative to the MEM values. Except for an area within the SNAT2 coding region, total H3 protein was largely unchanged by amino acid limitation (Figure 2b). These results demonstrate that there is region-specific chromatin remodeling and an active recruitment of the preinitiation complex to the SNAT2 promoter during amino acid stress.Figure 1.


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)

Amino acid limitation induces region-specific changes in histone acetylation, SNAT2 transcription activity and GTF binding at the SNAT2 gene. (a) The SNAT2 transcription activity and the binding of RNA Pol II, both published elsewhere (14), are provided as a graphical representation to provide a comparison with the ChIP data for the remaining GTFs. To obtain the data of (a), HepG2 cells were incubated in MEM (open circle) or MEM lacking histidine (closed circle) for 1 h, 2 h, 4 h or 8 h and protein association with the SNAT2 promoter was monitored by ChIP assay. For each of the GTF proteins shown in (a) binding to the SNAT2 AARE was also tested, but was not significantly different from the background value obtained with a nonspecific antibody. An example is illustrated by the data for TFIIB (b). Nuclear extracts were prepared and subjected to immunoblot analysis for several GTFs, those for TAFI, Pol II and TFIIB are shown as examples (c).
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Figure 1: Amino acid limitation induces region-specific changes in histone acetylation, SNAT2 transcription activity and GTF binding at the SNAT2 gene. (a) The SNAT2 transcription activity and the binding of RNA Pol II, both published elsewhere (14), are provided as a graphical representation to provide a comparison with the ChIP data for the remaining GTFs. To obtain the data of (a), HepG2 cells were incubated in MEM (open circle) or MEM lacking histidine (closed circle) for 1 h, 2 h, 4 h or 8 h and protein association with the SNAT2 promoter was monitored by ChIP assay. For each of the GTF proteins shown in (a) binding to the SNAT2 AARE was also tested, but was not significantly different from the background value obtained with a nonspecific antibody. An example is illustrated by the data for TFIIB (b). Nuclear extracts were prepared and subjected to immunoblot analysis for several GTFs, those for TAFI, Pol II and TFIIB are shown as examples (c).
Mentions: The SNAT2 gene has an AARE in intron 1 (nucleotides +709 to +717) that functions as an ATF4-responsive enhancer to activate transcription within 1 h after amino acid limitation and the transcription remains elevated for about 10–12 h (13,14). The time course of SNAT2 transcription activity and recruitment of RNA Pol II have been published previously (14), but for the purpose of comparison a graphical representation is shown in the first panel of Figure 1a. It has been suggested that in the absence of Mediator, the TFIID complex may provide the link between enhancer elements and RNA Pol II (4). For SNAT2, the activator ATF4 binds to the intronic AARE, but does not appear to interact directly with the promoter region (12). To provide mechanistic insight into assembly of the preinitiation complex following amino acid limitation, binding of the TFIIA, TFIIB, TFIID (TAF1 and TAF9) and TFIIE was measured by ChIP analysis (Figure 1a). Following amino acid removal, all of the GTFs exhibited increased binding to the SNAT2 promoter in a manner that qualitatively paralleled the enhancement in transcription activity. In contrast, as shown by the example of TFIIB (Figure 1b), none of the GTFs showed significant binding to the intronic region containing the SNAT2 AARE (TFIIA, TFIID and TFIIE, not shown). The stress-induced recruitment of the GTFs was not a consequence of a change in their absolute concentration, as illustrated by immunoblotting of nuclear extracts (Figure 1c). Increased acetylation of histone H3 (AcH3) is a general marker for induced gene expression. To monitor the localization of amino acid-dependent chromatin modification for the SNAT2 gene, ChIP analysis for AcH3 was performed at specific regions across the SNAT2 gene 8 h after amino acid limitation (Figure 2a). The SNAT2 promoter exhibited the largest increase in AcH3, but acetylation was also elevated at the AARE. In contrast, at regions only about 1 kb upstream or downstream within the gene, there was little or no increase in histone modification relative to the MEM values. Except for an area within the SNAT2 coding region, total H3 protein was largely unchanged by amino acid limitation (Figure 2b). These results demonstrate that there is region-specific chromatin remodeling and an active recruitment of the preinitiation complex to the SNAT2 promoter during amino acid stress.Figure 1.

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