PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary...

Area of Science:

• Developmental Biology
• Molecular Biology
• Genetics

Background:

• Sonic hedgehog (SHH) signaling is crucial for craniofacial morphogenesis.
• Pre-B-cell leukemia homeobox (PBX) transcription factors are implicated in midfacial development.
• The precise interaction between PBX factors and SHH regulation in facial development remains unclear.

Purpose of the Study:

• To investigate the role of PBX1 and PBX3 in regulating SHH expression within the frontonasal ectodermal zone (FEZ).
• To identify and characterize cis-regulatory elements involved in PBX-mediated SHH modulation.
• To elucidate the distinct functions of PBX1 and PBX3 in craniofacial development.

Main Methods:

• Perturbation of PBX1/3 expression in chick embryos using RCAS viruses.
• Assessment of SHH expression levels at specific developmental stages (HH10-HH22).
• Multi-omics approaches including ATAC-seq and ChIP-seq to map PBX binding and chromatin accessibility.
• Reporter assays (in ovo electroporation and in vitro luciferase assays) to validate enhancer activity.

Main Results:

• PBX1 overexpression expanded SHH expression, while PBX3 overexpression decreased it.
• Reduced PBX1 expression decreased SHH expression; reduced PBX3 expression induced ectopic SHH.
• A novel PBX1-enriched cis-regulatory element (SFE1) in the SHH intron was identified, exhibiting enhancer activity.
• PBX1 binding to SFE1 upregulated SHH transcription, whereas PBX3 binding downregulated it.

Conclusions:

• PBX1 and PBX3 play complementary and opposing roles in regulating SHH expression.
• The identified SFE1 element is a key cis-regulatory region interacting with PBX1/3 to modulate SHH.
• These findings provide critical insights into the molecular mechanisms governing craniofacial development.