Foxn3 is part of a transcriptional network that regulates primary cilia in the developing mouse retina [CUT & RUN]

Analysis of retinas from mice with a targeted disruption of the Foxn3 gene revealed additional displaced amacrine interneurons and additional retinal astrocytes in the inner plexiform layer and in the ganglion cell layer. The forkhead transcription factor Foxn3 is a transcriptional repressor. Numerous genes linked to cilia structure or assembly were upregulated in embryonic retinas without Foxn3, including Foxj1, a forkhead transcription factor required for motile cilia. Ectopic primary cilia were observed on bipolar and amacrine interneurons in postnatal retinas without Foxn3. CUT&RUN analysis revealed that many upregulated retinal genes were bound by the Foxn3 and Rfx3 proteins. A short hydrophobic motif shared by Foxj1, Foxn4, and Foxn3 proteins was found to be required for association with the Rfx3 protein and for transcriptional repression by Foxn3, as well as for full transcriptional activation by Foxn4 with Rfx3. AlphaFold 3 predicts an interaction between the hydrophobic motif and the Rfx3 dimerization domain. Mutations in Rfx3 at the predicted interaction site disrupted association of Rfx3 with Foxn3 or Foxn4. These results reveal a new layer of transcriptional regulation of genes required for cilia, with Foxn3 functioning as a repressor of cilia genes and limiting primary cilia formation in the developing retina. Overall design: CUT&RUN analysis of genomic binding sites for the Foxn3, Foxn4, and Rfx3 transcription factors in E16 CD-1 mouse retinas.