Part:BBa_K4414010

NR3C1

This basic part consists of the amino-terminal domain (NTD), DNA binding domain (DBD), hinge region and ligand binding domain (LBD)(BBa_K4414000).[1]

Usage and Biology

As glucocorticoid receptor, it can function both as a transcription factor that binds to glucocorticoid response elements (GRE) in the promoters of glucocorticoid responsive genes to activate their transcription, and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus.[2]

Figure1. Schematic figure of BBa_K4414010

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Validation

Method

1. HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414010 and SEAP with GRE3 or GRE6. Cells were treated with 10, 50, or 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [3]

Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-Tyr into cells

2. HEK-293T cells were transfected with plasmid encoding BBa_K4414010 and BBa_K1123017. Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. The fluorescence intensity of cells was observed 24 h after posting glucocorticoids treatment.

Result

Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1-2 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 2). The fluorescence microscopic image showed GR locates in the nucleus whether treated with glucocorticoids or not (Figure 3).

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.

Figure4. Fluorescence intensity of cells mediated by BBa_K4414010 and BBa_K1123017.

Reference

[1]Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982

[2]Lu NZ, Cidlowski JA. Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell. 2005 Apr 29;18(3):331-42. doi: 10.1016/j.molcel.2005.03.025. PMID: 15866175.

[3]Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021;2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.