Journal: Nature Communications

Article Title: Co-occupancy identifies transcription factor co-operation for axon growth

doi: 10.1038/s41467-021-22828-3

Figure Lengend Snippet: a Overview of sample collection for RNA-Seq analysis. (CSMNs, corticospinal motor neurons). b Nuclei before and after FANS, confirming successful purification of mNeongreen+ nuclei. c Venn diagram showing transcript comparison across the three gene treatments. d Regulatory network analysis of genes upregulated after Klf6 overexpression revealed subnetworks enriched for distinct functional categories highly relevant to axon growth. Nodes correspond to target genes and edges to multiple modes of interaction (physical, shared upstream regulators, shared signaling pathways, and inter-regulation). Only significantly enriched GO categories were included in the network analysis ( p < 0.05, right-sided hypergeometric test with Bonferroni correction, Cytoscape). e Heatmap showing selection of differentially expressed genes between control and Klf6 treatment. f The number of genes within each functional subnetwork (top) and the total FPKM values in each in control and Klf6 overexpression groups. g Regulatory network analysis of genes uniquely upregulated after combined Klf6 and Nr5a2 overexpression (not upregulated in single treatments) revealed subnetworks enriched for functions in macromolecule biosynthesis and DNA repair ( p < 0.05, right-sided hypergeometric test with Bonferroni correction, Cytoscape). h Heatmap showing selection of differentially expressed genes in DNA repair and macromolecule biosynthesis modules. Genes that are upregulated (green) and downregulated (red) relative to control are shown. Color values indicate average FPKM expression values as shown in the legend. Columns correspond to treatment groups and rows to genes. i The percent change in FPKM values within each module, revealing that combined Klf6 and Nr5a2 treatment most affect genes in DNA repair and macromolecule biosynthesis modules (top). Graph shows the number of genes within functional subnetworks (bottom). n = 3 animals pooled per replicate, 2 replicates/gene treatment with similar results (Supplementary Data ). Scale bar is 100 µm. Source data are provided as a Source Data file.

Article Snippet: Constructs for candidate genes were purchased from Dharmacon or Origene, and relevant accession numbers for all 62 candidate TFs are summarized in Supplementary Data . mScarlet was a gift from Dorus Gadella (RRID:Addgene_85044), pAAV-CAG-tdTomato (codon diversified) was a gift from Edward Boyden (RRID:Addgene_59462), and mNeonGreen sequences were synthesized by Genscript, based on the amino acid sequence of ref. . For viral production, genes were cloned into an AAV-CAG backbone (Addgene Plasmid #59462) using standard PCR amplification .

Techniques: RNA Sequencing, Purification, Comparison, Over Expression, Functional Assay, Protein-Protein interactions, Selection, Control, Expressing