Rfx2 Stabilizes Foxj1 Binding at Chromatin Loops to Enable Multiciliated Cell Gene Expression

Area of Science:

• Genomics
• Molecular Biology
• Developmental Biology

Background:

• Cooperative transcription factor binding is crucial for eukaryotic gene expression.
• Motile cilia formation requires Forkhead (Fox) and Rfx transcription factors, which co-localize to cilia gene promoters.
• The precise regulatory mechanisms and genomic context of these factors remain unclear.

Purpose of the Study:

• To investigate the genome-wide targets of Foxj1 and Rfx2 in cilia gene regulation.
• To elucidate the direct vs. indirect binding mechanisms of Foxj1 and Rfx2.
• To understand how these factors function within the 3D genome architecture.

Main Methods:

• Genome-wide approaches to identify cis-regulatory sites.
• Analysis of transcription factor binding (Foxj1, Rfx2) at promoters and enhancers.
• Chromatin conformation capture (3C) to identify looping interactions.

Main Results:

• Cilia genes are located at topological domain boundaries with low enhancer density.
• Foxj1 and Rfx2 bind to more cilia gene promoters than other known factors.
• Rfx2 binds promoters and enhancers equally, while Foxj1 prefers enhancers and is stabilized by Rfx2 at promoters.
• Rfx2 and Foxj1 anchor chromatin loops, suggesting Rfx2 facilitates distal enhancer-promoter communication.

Conclusions:

• Rfx2 acts as a scaffolding factor, stabilizing distal enhancers and proximal promoters.
• This scaffolding function brings regulatory domains into proximity, enabling coordinated cilia gene expression.
• Foxj1 and Rfx2 cooperatively regulate motile cilia formation through 3D genome organization.