The mineralocorticoid receptor forms higher order oligomers upon DNA binding
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Abstract
The prevailing model of steroid hormone nuclear receptor function assumes ligand‐induced homodimer formation followed by binding to DNA hormone response elements (HREs). This model has been challenged by evidence showing that the glucocorticoid receptor (GR) forms tetramers upon ligand and DNA binding, which then drive receptor‐mediated gene transactivation and transrepression. GR and the closely‐related mineralocorticoid receptors (MR) interact to transduce corticosteroid hormone signaling, but whether they share the same quaternary arrangement is unknown. Here, we used a fluorescence imaging technique, Number & Brightness, to study oligomerization in a cell system allowing real‐time analysis of receptor‐DNA interactions. Agonist‐bound MR forms tetramers in the nucleoplasm and higher order oligomers upon binding to HREs. Antagonists form intermediate‐size quaternary arrangements, suggesting that large oligomers are essential for function. Divergence between MR and GR quaternary structure is driven by different functionality of known and new multimerization interfaces, which does not preclude formation of heteromers. Thus, influencing oligomerization may be important to selectively modulate corticosteroid signaling.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8072766.
Summary:
This paper aims to answer the question of the oligomeric states of the mineralocorticoid (MR) in the nucleus and when bound to DNA hormone response elements (HREs) in vivo. Using Number & Brightness (N&B) analysis, they investigate the oligomeric state of MR in the presence of different ligands and mutations/truncations to identify what controls different oligomeric states. While they comparisons they performed between different ligands and constructs of MR show qualitative differences, this paper is missing some key controls, particularly in the localization of the nucleus and MMTV array, which prevent us from thoroughly assessing the paper.
Major Comments:
1) Provide …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8072766.
Summary:
This paper aims to answer the question of the oligomeric states of the mineralocorticoid (MR) in the nucleus and when bound to DNA hormone response elements (HREs) in vivo. Using Number & Brightness (N&B) analysis, they investigate the oligomeric state of MR in the presence of different ligands and mutations/truncations to identify what controls different oligomeric states. While they comparisons they performed between different ligands and constructs of MR show qualitative differences, this paper is missing some key controls, particularly in the localization of the nucleus and MMTV array, which prevent us from thoroughly assessing the paper.
Major Comments:
1) Provide details and controls on identifying the nucleus versus cytoplasmic versus DNA binding/MMTV array. Only labeling these with the molecule of interest (MR) is inappropriate. To assess if MR is congregating at the MMTV or some other location in the nucleus, the MMTV array must be labeled with something other than GFP, allowing simultaneous visualization of the array and the MR oligomerization state.
2) Explain the varying oligomerization states you observe across your dataset. Can you provide ranges of oligomerization states across your results? How should we interpret mixed populations? What were your criteria to decide whether a construct dimerizes, oligomerizes etc.
3) The manuscript has varying points for each condition (for example, in Figure 1B, there are 490 single cells for one condition, with 36 single cells for another condition). Please explain why there is so much variety in the number of data points.
Minor Comments:
1) The introduction could be improved by expanding on details on the transcriptional crosstalk of MR/GR and the observations of GR at MMTV (and clarify this is the data the rest of this paper is compared to).
2) Please clarify the construct of the cell line and if endogenous MR is knocked out.
3) In Fig. 4 C, the MR-N579/GC-470C mutant array displays only 11 data points, while the figure legend says it contains 22.
4) The figures with agonist or antagonist would be clearer if the agonist or antagonists were labeled.
5) In the section 'MR and GR do not share the same dimerization interfaces'. Please provide some context for the D-loop and P-loop. Figure 3A could be improved by showing where these are structurally or among the entire sequence.
6) Please specify how many independent experiments were run for each condition.
8) The authors describe that imaging happened 30min - 2h after ligand adding. Please specify what experiment was incubated with ligand and for how long. Is it possible that the signal is increasing proportionally with longer incubation times? A comparison in the Supplementary would be helpful.
Competing interests
The author declares that they have no competing interests.
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