The Glucocorticoid Receptor is Required for Efficient Aldosterone-Induced Transcription by the Mineralocorticoid Receptor.

The glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) have distinct, yet overlapping physiological and pathophysiological functions. There is strong indication that both receptors interact both functionally and physically, but the precise role of this interdependence is poorly understood. Here, we analyzed the impact of GR co-expression on MR genome-wide chromatin binding and transcriptional responses to aldosterone and glucocorticoids, both physiological ligands of this receptor. Our data show that GR co-expression alters MR genome-wide binding in a locus- and ligandspecific way. MR binding to consensus DNA sequences is affected by GR. Transcriptional responses of MR in the absence of GR are weak and show poor correlation with chromatin binding. In contrast, coexpression of GR potentiated MR-mediated transcription, particularly in response to aldosterone. Finally, single-molecule tracking of MR suggests that the presence of GR contributes to productive binding to chromatin. Together, our data indicate that co-expression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids

The mineralocorticoid receptor forms higher order oligomers upon DNA binding

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The mineralocorticoid receptor forms higher order oligomers upon DNA binding.

The mineralocorticoid and glucocorticoid receptors (MR and GR) are evolutionary related nuclear receptors with highly conserved DNA- and ligand-binding domains (DBD and LBD), which determine promiscuous activation by corticosteroid hormones (aldosterone and glucocorticoids) and binding to a shared DNA consensus sequence, the hormone response element (HRE). In addition, MR and GR functionally interact, likely through direct formation of heteromeric complexes, potentially contributing to cell-specific corticosteroid signaling. It has recently been proposed that agonist and DNA binding promote GR self-association in tetramers. Here we investigated MR quaternary arrangement after receptor activation. To that end we used a fluorescence imaging technique, Number & Brightness (N&B) analysis, in a cell system where receptor-DNA interaction can be studied in live cells in real time. Our results show that agonist-bound MR is a tetramer in the nucleoplasm, forming higher order oligomers upon binding to HREs. Antagonists form intermediate quaternary arrangements, suggesting that the formation of large oligomeric complexes is essential for function. We also show that divergence between MR and GR quaternary arrangements are driven by different functionality of multimerization interfaces in the DBD and LBD and their interplay with the N-terminal domain. In spite of contrasting quaternary structures, MR and GR are able to form heteromers. Given the importance of both receptors as pharmacological targets and the differential oligomerization induced by antagonists, our findings suggest that influencing quaternary structure may be important to provide selective modulation of corticosteroid signaling

Single-molecule tracking reveals two low-mobility states for chromatin and transcriptional regulators within the nucleus

peer reviewedHow transcription factors (TFs) navigate the complex nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Using single-molecule tracking, coupled with machine learning, we examined the mobility of multiple transcriptional regulators. We show that H2B and ten different transcriptional regulators display two distinct low-mobility states. Our results indicate that both states represent dynamic interactions with chromatin. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that only chromatin interactions in the lowest mobility state require an intact DNA-binding domain as well as oligomerization domains. Importantly, these states are not spatially separated as previously believed but in fact, individual H2B and TF molecules can dynamically switch between them. Together, our results identify two unique and distinct low-mobility states of transcriptional regulators that appear to represent common pathways for transcription activation in mammalian cells

Mechanistic characterization of promoter/enhancer progression for glucocorticoid receptor responsive genes

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Power-law behavior of Transcription Factors dynamic

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Cell death signaling in cancer cells treated by Photodynamic Therapy

Glioblastoma multiformes (GBMs) are an extremely aggressive and infiltrating type of brain cancer. Despite heavy therapies and extensive fundamental and applied research, the median survival of patients remains about 15 months after diagnosis for over a decade. Therefore, there is an emergency to find new approaches and therapeutic targets for treating this cancer. Photodynamic therapy (PDT) recently demonstrated a high potential in the treatment of GBM. Moreover, GBMs induced cell death by PDT is dependent of an atypical RIP3-dependent programmed necrosis. In parallel, GBMs activate a pro-survival autophagic pathway in order to recycle PDT-damaged structures and organelles. In our PhD thesis, we investigated the regulation of this autophagic process and found that TSC2 protein had an important role in autophagy activation by PDT. Indeed, PDT treatment quickly activates the kinase MK2, which phosphorylates TSC2 on serine 1254. We then showed that phosphorylation of this serine was crucial for autophagy activation, which makes TSC2 a crucial pro-survival factor in GBMs treated with PDT. Finally, we demonstrated that protein 14-3-3 ζ (YWHAZ) interacts with TSC2 and protects TSC2 serine 1254 phosphorylation from phosphatase actions after PDT. In the same time we conducted a proteomic analysis on RIP3 immunoprecipitate. The major implication of this analysis is the demonstration that RIP3 interacts with TSC2 and YWHAZ. Finally, we showed that RIP3 interacts better with the non-phosphorylated form of TSC2 than with the phosphorylated form, suggesting a RIP3 interference in the TSC2-dependent autophagy activation process. These data were submitted for publication in "Scientific Reports". In the second part of this thesis, we also investigated the influence of RIP3 expression on osteosarcoma (U2OS) cell death. U2OS expressing or not RIP3 were treated with PDT and cell death mechanisms have been investigated. We first demonstrated that in both cell lines, apoptosis was the major cell death mechanism. Secondly, we noticed an over-activation of various caspases in cells expressing RIP3 despite a stronger resistance to PDT. This could be explained by a lower activation of autophagy in cells not expressing RIP3. Thirdly, we showed that in RIP3 expressing cells, residual necrosis was RIP1-dependent. We therefore suggest that RIP3 is able to influence the cell death process. These data were published in "Laser in surgery and medicine."Etude du signaling de mort induit dans les glioblastomes par la thérapie photodynamiqu

Toward A Mechanistic Characterization of Promoter Progression for Glucocorticoid Receptor Responsive Genes

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