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. 2007;35(1):35-44.
doi: 10.1093/nar/gkl987. Epub 2006 Dec 5.

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

Lin Ma  1 , Yuk Y Sham, Kylie J Walters, Howard C Towle
Affiliations

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

"VSports app下载" Lin Ma et al. Nucleic Acids Res. 2007.

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 VSports手机版. 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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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Mutational analysis of ChREBP/Mlx binding to the PK ChoRE. (a) Sequences of probes used to analyze ChREBP/Mlx binding. Probe A is the wild-type PK ChoRE and probe F is the synthetic WBSCR14 probe. Mutations from the wild-type PK ChoRE in probes B through E are shown in rectangles. The E box-like sequence of the PK probe and the corresponding region of mutant probes are capitalized, as is the single E box of the WBSCR14 probe. (b) EMSA was performed with probes shown in part (a). All odd-numbered lanes contain 5 μg of mock-transfected 293 whole cell extract and even-numbered lanes contain 5 μg of whole cell extract from 293 cells transfected with ChREBP and Mlx. The solid arrow indicates the slower migrating ChREBP/Mlx complex, and the open arrow the faster migrating complex. Asterisks denote background binding.
Figure 3
Figure 3
Model of the bHLH/LZ region of two ChREBP/Mlx heterodimers binding to the ChoRE. The models were generated by applying energy minimization and molecular dynamics (for the loop regions) on homology-based structures. The helical regions of Mlx and ChREBP are indicated in red and yellow, respectively, whereas the E boxes and flanking DNA are highlighted in blue and black. The three residues that were hypothesized and confirmed to contribute to ChoRE binding are highlighted in purple: F164, I166 and K170. Molecular modeling was performed on complexes in which the Mlx loop of one heterodimer partner is oriented toward the Mlx loop of the other partner (a) or toward the ChREBP loop of the other partner (b).
Figure 4
Figure 4
The Mlx loop region is critical for ChoRE binding. (a) Comparison of loop regions of various bHLH/LZ proteins. Sequences of the loop regions of several representative bHLH/LZ proteins are shown. The number of residues in the loop region is indicated. Mlx has a significantly longer loop domain than most other bHLH/LZ proteins, allowing it to potentially interact across the interface between heterodimer pairs. (b) Construction of ChREBP and Mlx loop variants. ChREBP loop variant: the loop region of ChREBP was substituted with the MondoA loop region; Mlx loop variant: the loop region of Mlx was substituted with the MondoA loop region. (c) EMSAs were performed with an oligonucleotide containing the PK ChoRE or the WBSCR14 probe and 5 μg of 293 cell extract. Lanes 1 and 8 are 293 mock-transfected whole cell extract. The other lanes contain extract from 293 cells transfected with the following expression plasmids: lanes 2 and 9, ChREBP loop variant alone; lanes 3 and 10, Mlx loop variant alone; lanes 4 and 11, ChREBP and Mlx; lanes 5 and 12, ChREBP and Mlx loop variant; lanes 6 and 13, ChREBP loop variant and Mlx; lanes 7 and 14, ChREBP loop variant and Mlx loop variant. The solid arrow indicates the slower migrating and the open arrow indicates the faster migrating ChREBP/Mlx complexes. Asterisks denote background binding. (d) Protein levels of ChREBP and Mlx in 293 cell extracts. Loading is as follows: lane 1, ChREBP and Mlx; lane 2, ChREBP and Mlx loop variant; lane 3, ChREBP loop variant and Mlx; lane 4, ChREBP loop variant and Mlx loop variant. Proteins were separated on an SDS–PAGE gel and immunoblotted with a ChREBP antibody to detect ChREBP and ChREBP loop variant (left side) or an anti-HA antibody to detect Mlx and Mlx loop variant (right side).
Figure 5
Figure 5
The loop region of Mlx is critical for functional activity. (a) Mlx function was measured by its ability to rescue a glucose-response in cells treated with a dominant negative Mlx adenovirus. Hepatocytes were treated with the dominant negative Mlx virus or control virus for 2 h. Plasmids expressing Mlx or the Mlx loop variant (50, 100 and 500 ng) were cotransfected into hepatocytes together with a reporter containing multiple copies of the ACC ChoRE linked to a basal promoter. Cells were incubated in low glucose for 24 h and then changed to either low or high glucose for an additional 24 h to induce the glucose response. Values represent the mean of triplicate samples (±SD). (b) Same as (a) except the reporter contained the PK promoter fragment from −183 to +12. (c) ChREBP function was measured in an identical manner to that described for Mlx in (a) except a dominant negative ChREBP adenovirus was used in place of the dominant negative Mlx virus.
Figure 6
Figure 6
Three residues in the loop region are critical for Mlx binding and function. A mutant form of Mlx in which three loop residues—F164, I166 and K170—were changed to alanine was constructed. (a) EMSA was performed with an oligonucleotide containing the ACC ChoRE (5) or the WBSCR14 probe. Lanes 1 and 4 are the negative controls of mock-transfected 293 whole cell lysate. Lanes 2 and 5 are 293 cell whole cell extracts expressing both ChREBP and Mlx. Lanes 3 and 6 are ChREBP and Mlx loop mutant co-expressed in 293 cells. The solid arrow indicates the slower migrating and the open arrow the faster migrating ChREBP/Mlx complexes. Asterisks indicate background binding. (b) Protein levels of wild-type and mutant Mlx (left) and ChREBP (right). The loading corresponds to the loading used in the EMSA in panel (a). Proteins were separated on a SDS–PAGE gel and immunoblotted with an anti-HA antibody to detect wild-type and loop mutant Mlx, while an anti-FLAG antibody was used to detect ChREBP. (c) Hepatocytes were treated with dominant negative Mlx virus or control virus for 2 h. Increasing amounts of plasmids (50, 100 and 500 ng) expressing wild-type Mlx or Mlx variant (F, I, K/A) were cotransfected with the ACC ChoRE-containing reporter into hepatocytes. Cells were incubated in low glucose for 24 h and then changed to either low or high glucose for an additional 24 h to induce the glucose response. Values represent the mean of triplicate samples (±SD).
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"VSports注册入口" References

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