Human-zebrafish non-coding conserved elements act in vivo to regulate transcription.

Area of Science:

• Genomics
• Evolutionary Biology
• Molecular Biology

Background:

• Comparative genomics aids in identifying functionally important genomic sequences like core genes and cis-acting regulatory elements (REs).
• Experimental validation is crucial to confirm the biological activity of predicted regulatory elements (REs) from non-coding (NC) sequences.

Purpose of the Study:

• To assess the effectiveness of comparative genomics in identifying active REs from anonymous NC sequences.
• To develop and validate novel in vivo assays for testing the transcriptional regulatory potential of candidate REs.

Main Methods:

• Generated a novel alignment of human and zebrafish genomes, contrasted with human and fugu datasets.
• Utilized zebrafish transient transgenesis to test 16 candidate REs for cis-acting gene regulatory properties using a green fluorescent protein (GFP) reporter.
• Developed a quantitative enhancer assay based on normalized luciferase activity for high-throughput screening.

Main Results:

• Identified 1744 core vertebrate REs by selecting deeply conserved non-genic elements across human and two fish genomes.
• In vivo testing confirmed cis-acting function in 10 out of 16 (63%) candidate REs, modulating tissue-specific gene expression.
• The novel luciferase assay identified 11 out of 16 (69%) REs with cis-acting function, including 9 previously confirmed by GFP assay.

Conclusions:

• Comparative genomics of distantly related vertebrate species is a valuable tool for identifying functional REs.
• The developed quantitative in vivo enhancer assay offers a scalable method for defining the functional activity of candidate REs.