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A critical role for the loop region of the basic helix-loop-helix/leucine zipper protein Mlx in DNA binding and glucose-regulated transcription.

Ma L, Sham YY, Walters KJ, Towle HC - Nucleic Acids Res. (2006)

Bottom Line: The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose.These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity.In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix-loop-helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

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Comparison of binding of ChREBP/Mlx to oligonucleotides containing a single E box or the PK ChoRE. EMSA was performed with a probe containing the ChoRE of the PK gene, which consists of two E box-like motifs (5), or a probe containing only one perfect E box sequence (WBSCR14) (23). Sequences of these oligonucleotides are shown in Figure 2. Lanes 1 and 8 are controls with 5 μg of mock-transfected 293 whole cell extract. The remaining lanes are loaded with increasing amounts (0.5–5 μg) of whole cell extract from 293 cells co-expressing ChREBP and Mlx. The solid arrow indicates the slower migrating ChREBP/Mlx complex. The open arrow indicates the faster migrating ChREBP/Mlx complex. Asterisks denote background binding.
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fig1: Comparison of binding of ChREBP/Mlx to oligonucleotides containing a single E box or the PK ChoRE. EMSA was performed with a probe containing the ChoRE of the PK gene, which consists of two E box-like motifs (5), or a probe containing only one perfect E box sequence (WBSCR14) (23). Sequences of these oligonucleotides are shown in Figure 2. Lanes 1 and 8 are controls with 5 μg of mock-transfected 293 whole cell extract. The remaining lanes are loaded with increasing amounts (0.5–5 μg) of whole cell extract from 293 cells co-expressing ChREBP and Mlx. The solid arrow indicates the slower migrating ChREBP/Mlx complex. The open arrow indicates the faster migrating ChREBP/Mlx complex. Asterisks denote background binding.

Mentions: All ChoREs studied to date contain two E box-like motifs separated by 5 bp (22). Previous results indicate that ChREBP and Mlx bind the ChoRE as a complex requiring both proteins (17). Interestingly, ChREBP and Mlx also bind as a complex to oligonucleotides containing only a single E box, as long as this site contains a perfect CACGTG consensus sequence (17,23). This complex migrates more rapidly in an EMSA than that formed on several ChoREs (17). We surmised that each of the E box-like motifs of the ChoRE binds to a ChREBP/Mlx heterodimer, which would cause such complexes to migrate more slowly in an EMSA compared to a single ChREBP/Mlx heterodimer formed with only one E box motif. To test whether binding of ChREBP/Mlx to the ChoRE is dependent on interactions between heterodimers bound at the tandemly arranged E box-like motifs, we performed an EMSA experiment with various amounts of overexpressed ChREBP/Mlx proteins and either an oligonucleotide containing a single perfect E box motif (WBSCR14) or the PK ChoRE oligonucleotide with two degenerate E boxes (Figure 1). Lanes 1 and 8 are the negative controls of mock-transfected 293 whole cell extract corresponding to the highest amount of protein used in the titration. All bands in these lanes represent background binding, as ChREBP and Mlx are expressed at negligible levels in 293 cells. The remaining lanes are loaded with increasing amounts of whole cell extract from 293 cells co-expressing a S196A/T666A double mutant ChREBP and wild-type Mlx. The double mutant ChREBP was used here to enhance the binding of ChREBP/Mlx to the ChoRE sequence (18,24). As reported previously (17), a single complex was observed on both PK ChoRE and WBSCR14 oligonucleotides. However, the PK ChoRE complex migrated more slowly than the WBSCR14 complex. Only the slower migrating complex appeared on the PK ChoRE even at the lowest amount of ChREBP/Mlx protein. If the binding of ChREBP/Mlx heterodimers to the two E box-like motifs were independent of each other, then at low ratios of protein to DNA, the faster migrating population should appear. Therefore, we hypothesized that two ChREBP/Mlx heterodimers are required to stabilize the binding of Mlx/ChREBP to the tandemly arranged E box-like motifs of the ChoRE.


A critical role for the loop region of the basic helix-loop-helix/leucine zipper protein Mlx in DNA binding and glucose-regulated transcription.

Ma L, Sham YY, Walters KJ, Towle HC - Nucleic Acids Res. (2006)

Comparison of binding of ChREBP/Mlx to oligonucleotides containing a single E box or the PK ChoRE. EMSA was performed with a probe containing the ChoRE of the PK gene, which consists of two E box-like motifs (5), or a probe containing only one perfect E box sequence (WBSCR14) (23). Sequences of these oligonucleotides are shown in Figure 2. Lanes 1 and 8 are controls with 5 μg of mock-transfected 293 whole cell extract. The remaining lanes are loaded with increasing amounts (0.5–5 μg) of whole cell extract from 293 cells co-expressing ChREBP and Mlx. The solid arrow indicates the slower migrating ChREBP/Mlx complex. The open arrow indicates the faster migrating ChREBP/Mlx complex. Asterisks denote background binding.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC1761440&req=5

fig1: Comparison of binding of ChREBP/Mlx to oligonucleotides containing a single E box or the PK ChoRE. EMSA was performed with a probe containing the ChoRE of the PK gene, which consists of two E box-like motifs (5), or a probe containing only one perfect E box sequence (WBSCR14) (23). Sequences of these oligonucleotides are shown in Figure 2. Lanes 1 and 8 are controls with 5 μg of mock-transfected 293 whole cell extract. The remaining lanes are loaded with increasing amounts (0.5–5 μg) of whole cell extract from 293 cells co-expressing ChREBP and Mlx. The solid arrow indicates the slower migrating ChREBP/Mlx complex. The open arrow indicates the faster migrating ChREBP/Mlx complex. Asterisks denote background binding.
Mentions: All ChoREs studied to date contain two E box-like motifs separated by 5 bp (22). Previous results indicate that ChREBP and Mlx bind the ChoRE as a complex requiring both proteins (17). Interestingly, ChREBP and Mlx also bind as a complex to oligonucleotides containing only a single E box, as long as this site contains a perfect CACGTG consensus sequence (17,23). This complex migrates more rapidly in an EMSA than that formed on several ChoREs (17). We surmised that each of the E box-like motifs of the ChoRE binds to a ChREBP/Mlx heterodimer, which would cause such complexes to migrate more slowly in an EMSA compared to a single ChREBP/Mlx heterodimer formed with only one E box motif. To test whether binding of ChREBP/Mlx to the ChoRE is dependent on interactions between heterodimers bound at the tandemly arranged E box-like motifs, we performed an EMSA experiment with various amounts of overexpressed ChREBP/Mlx proteins and either an oligonucleotide containing a single perfect E box motif (WBSCR14) or the PK ChoRE oligonucleotide with two degenerate E boxes (Figure 1). Lanes 1 and 8 are the negative controls of mock-transfected 293 whole cell extract corresponding to the highest amount of protein used in the titration. All bands in these lanes represent background binding, as ChREBP and Mlx are expressed at negligible levels in 293 cells. The remaining lanes are loaded with increasing amounts of whole cell extract from 293 cells co-expressing a S196A/T666A double mutant ChREBP and wild-type Mlx. The double mutant ChREBP was used here to enhance the binding of ChREBP/Mlx to the ChoRE sequence (18,24). As reported previously (17), a single complex was observed on both PK ChoRE and WBSCR14 oligonucleotides. However, the PK ChoRE complex migrated more slowly than the WBSCR14 complex. Only the slower migrating complex appeared on the PK ChoRE even at the lowest amount of ChREBP/Mlx protein. If the binding of ChREBP/Mlx heterodimers to the two E box-like motifs were independent of each other, then at low ratios of protein to DNA, the faster migrating population should appear. Therefore, we hypothesized that two ChREBP/Mlx heterodimers are required to stabilize the binding of Mlx/ChREBP to the tandemly arranged E box-like motifs of the ChoRE.

Bottom Line: The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose.These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity.In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix-loop-helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

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