Abstract
Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRβ, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue.
Highlights
Appropriate levels of Thyroid hormone (TH) are critical for the proper development and maturation of several tissues, including the brain [1,2,3,4,5,6,7,8], cochlea [9,10,11,12,13], and retina [14,15,16,17]
To investigate the ability of each thyroid response elements (TREs) to positively respond to T3 in the context of a minimal TATA, each construct was transfected into 293T cells and cultured 6T3
Each TRE was qualitatively assayed for its ability to promote green fluorescent protein (GFP) and alkaline phosphatase (AP) activity in response to T3 addition, or repress GFP and AP activity in the absence of exogenous T3, to verify that these previously validated T3-responsive TREs [22,59,60,61,62,63,64,65,66,67] were still able to promote T3-dependent activity using only a minimal TATA
Summary
Appropriate levels of TH are critical for the proper development and maturation of several tissues, including the brain [1,2,3,4,5,6,7,8], cochlea [9,10,11,12,13], and retina [14,15,16,17]. TRs are nuclear hormone transcription factors that bind specific DNA sequences (TREs) and modulate gene transcription in a ligand-dependent bimodal fashion. Heterodimerization with a retinoid X receptor (RXR) enhances binding of some nuclear receptors (NRs), including the Vitamin D3 Receptors (VDRs), TRs, and Retinoic Acid Receptors (RARs), to their respective response elements [18,19,20]. Ligand-specificity is conferred via the spacing and orientation of each half site repeat. One well characterized T3-specific TRE is the direct repeat +4 element (DR4), consisting of the sequence AGGTCAnnnnAGGTCA. Adding or subtracting one base from the interval between the two direct repeats diminishes T3 ligand sensitivity and introduces sensitivity to either retinoic acid (RA) with a 5 nucleotide spacer (DR+5) or Vitamin D3 (VitD3) with a 3 nucleotide spacer (DR+3) [22]
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