Functional Definition of Thyroid Hormone Response Elements Based on a Synthetic STARR-seq Screen.

Area of Science:

• Molecular Biology
• Genetics
• Endocrinology

Background:

• The nuclear receptor TRα1 regulates gene transcription upon binding thyroid hormone.
• TRα1 functions as a heterodimer with retinoid X receptors, targeting DR4 consensus sequences.
• Genomic DR4-like elements vastly outnumber occupied sites, suggesting sequence nuances dictate TRα1 activity.

Purpose of the Study:

• To investigate how minor nucleotide variations in DNA response elements affect TRα1 DNA-binding and transactivation.
• To quantitatively assess the transcriptional activity of numerous synthetic DNA sites in parallel.
• To establish the relationship between DNA-binding affinity and thyroid hormone response mediated by TRα1 heterodimers.

Main Methods:

• Utilized an improved protocol for synthetic self-transcribing active regulatory region sequencing.
• Performed a functional screen to quantitatively assess transcriptional activity across thousands of synthetic DNA sites.
• Analyzed the correlation between DNA-binding affinity of TRα1-retinoid X receptor heterodimers and their transactivation capacity.

Main Results:

• Identified a strong correlation between the affinity of TRα1 heterodimers for specific DNA sequences and their ability to mediate thyroid hormone response.
• Demonstrated that subtle nucleotide variations significantly influence TRα1's DNA-binding capacity and transactivation activity.
• Quantitatively mapped the transcriptional activity of thousands of synthetic regulatory regions.

Conclusions:

• DNA sequence composition is a critical determinant of TRα1-mediated gene transcription and thyroid hormone signaling.
• The affinity of TRα1 heterodimers for DNA directly correlates with their functional role in thyroid hormone response.
• This study provides a quantitative framework for understanding sequence-specific gene regulation by nuclear receptors.