Using a modular massively parallel reporter assay to discover context-dependent regulatory activity in type 2...

Area of Science:

• Genomics
• Molecular Biology
• Endocrinology

Background:

• Complex traits are linked to noncoding genome regions, making functional annotation difficult.
• Massively parallel reporter assays (MPRAs) are used to test variant regulatory effects but often lack cell-type specificity.
• Understanding context-specific regulatory element function is crucial for interpreting genetic associations.

Purpose of the Study:

• To investigate the context-sensitivity of MPRAs by evaluating regulatory element activity in a pancreatic beta cell model.
• To assess the impact of MPRA construct design, including fragment orientation and promoter choice, on measured regulatory activity.
• To identify sequence features and transcription factor binding motifs associated with cell-specific regulatory activity relevant to metabolic traits.

Main Methods:

• Screened nearly 12,000 DNA fragments from diabetes-associated regions using MPRAs in a pancreatic beta cell line.
• Compared MPRA activity with fragments cloned upstream or downstream of a reporter gene.
• Tested fragments with both a synthetic housekeeping promoter (SCP1) and the human insulin gene promoter (INS).

Main Results:

• MPRA construct design significantly affected measured regulatory activity, with notable positional and promoter-specific biases observed.
• A majority of fragments showed preference for the cell-specific INS promoter over the housekeeping promoter.
• HNF1 binding motifs were enriched in fragments showing INS promoter preference, suggesting a role in beta cell regulation.
• Follow-up experiments confirmed HNF1-dependent regulation in beta cells but not in skeletal muscle cells.

Conclusions:

• MPRA results are sensitive to experimental design choices, including promoter selection and fragment orientation.
• Cell-specific promoters, like INS, are critical for uncovering biologically relevant regulatory activity in MPRA studies.
• HNF1 transcription factors play a significant role in regulating genes in pancreatic beta cells, linking MODY and T2D genetic factors.
• Careful consideration of technical factors is essential for robust MPRA experimental design and interpretation in complex trait research.