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| Author(s): |
Izabella Luisa Tambones
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2020-08-05 |
| Examining board members: |
Ana Carolina Migliorini Figueira;
Celso Eduardo Benedetti;
Fernando Alves de Melo
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| Advisor: | Ana Carolina Migliorini Figueira |
| Abstract | |
The Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is identified as an important element involved in the heart development context. Its participation in this biological context occurs by the regulation of specific genes, such as the slow myosin heavy chain (SMyHC3). The SMyHC3 is a quail atrium-specific gene, and its promoter is essential for the differentiation of cardiac chambers in different species. This atrium-specific activity is potentially regulated via a complex nuclear receptor response element (ECRRN) located in its promoter region. Cellular assays revealed that COUP-TFII is the main candidate for regulation of the SMyHC3 gene promoter, and suggest that its regulatory activity is performed in cooperation with androgen (AR) and glucocorticoid (GR) receptors. Thus, our objective was to evaluate the AR, GR and COUP-TFII interaction with each other and with the ECRRN seeking to understand the role of these receptors in the cardiac development regulation via the SMyHC3 promoter. Despite this, the AR protein was not included in our analyzes due to the difficulties in its production. Therefore, fluorescence anisotropy assays showed a greater affinity of COUP-TFII for ECRRN, compared to GR. Affinity measures were also performed between the receptors and the putative binding sites in the ECRRN (A, B, and C sites) and their combinations (AB, BC, and AC). Both receptors showed a preference for the same single site (B) and a double site (AB for COUP-TFII and BC for GR). Pull-down and microscale thermophoresis tests indicate that GR and COUP-TFII receptors interact physically (Kd = 3.4 ± 0.7 µM), and may form an atypical heterodimer. Computational analyzes by molecular docking and dynamics indicated a double interface between the hinge and DBD (1) and the LBDs (2) of GR and COUP-TFII. Cellular transactivation assays revealed a repressive crosstalk between GR and COUP-TFII in the regulation of DR1 and ECRRN responsive elements. Fluorescence anisotropy assays using both receptors and different response elements indicate that the transrepression between the NRs seen for the DR1 occurs by the coupling of GR in COUP-TFII. Finally, we confirm the crosstalk between GR and COUP-TFII in the regulation of the ECRRN. More specifically this crosstalk can occur directly, by coupling or the formation of the GR/COUP-TFII heterodimer or indirectly, via chromatin modification (AU) | |
| FAPESP's process: | 18/02481-0 - Regulation of the SMyHC iii promoter by the nuclear receptors AR, GR and Coup-TFII: a solo or joint work? |
| Grantee: | Izabella Luisa Tambones |
| Support Opportunities: | Scholarships in Brazil - Master |