Fig. 2: Evf2 guidance of Dlx5/6UCE to gene bodies (±5 kb) divides chr6 into transcriptionally activated (short-range) and repressed (long/super-long-range) domains, altering SMARCC2 and NONO binding at Dlx5/6UCE looping-regulated sites. | Nature Communications

Fig. 2: Evf2 guidance of Dlx5/6UCE to gene bodies (±5 kb) divides chr6 into transcriptionally activated (short-range) and repressed (long/super-long-range) domains, altering SMARCC2 and NONO binding at Dlx5/6UCE looping-regulated sites.

From: Single-cell transcriptomics of ventral forebrain progenitors identifies Evf2 enhancer lncRNA-enhancer gene guidance through direct RNA binding and RNP recruitment domains

Fig. 2: Evf2 guidance of Dlx5/6UCE to gene bodies (±5 kb) divides chr6 into transcriptionally activated (short-range) and repressed (long/super-long-range) domains, altering SMARCC2 and NONO binding at Dlx5/6UCE looping-regulated sites.

Differential scRNAseq analysis of Evf2-regulated genes located on chr6 (Evf2TS/TS÷ Evf2+/+) identifies cell type specificity, represented according to color (VZ, SVZ, Isl1, and Lhx6). A 15 Evf2 target gene locations on chr6 from 3.5 Mb (Ccdc132) −134Mb (Etv6), vertically represented with strand information (+) or (-), colored boxes on the left corresponding to cell types, Dlx5/6UCE locations within ±5 kb of the gene body (GB) indicated, with Evf2 guidance of Dlx5/6UCE to 13/15 GBs. 3D-Dlx5/6UCE positional shifts relative to GBs in the presence (Evf2+/+, pink boxes) and absence (Evf2TS/TS, blue boxes) of Evf2, co-regulated by SOX2 (bi-colored boxes), or gray boxes (independent of Evf2), genes associated with enhancers, E, or an ultraconserved sequence, U (Foxp2). Chr6 qA1/A2 (dashed line) division of chr6 into short-range and long-range genes, decrease (dotted arrow) and increase (gray) in Evf2TS/TS; average fold change (FC). B Previous proteomics sequencing of Evf2 RNP87 (yellow circle) identified diverse RNA binding proteins (dashed lines) and protein-protein interactions (dotted lines), also identifying SMARCC2 and NONO as Evf2 RNPs32, schematic modified from ref. 4. C Violin Plots compare Evf2 regulated RNP binding at Evf2 (+) and (-) regulated Dlx5/6UCE sites in E13.5GEs (Evf2TS/TS vs. Evf2+/+) (n = 2 libraries sequenced of 4 independent biological replicates of biologically pooled 6-8 E13.5GEs for each genotype, and each antibody, IgG subtracted). Cut&Run ≤120 bp fragments (directly bound) and ≥150 bp fragments (indirectly bound) profiles of SMARCC2 and NONO peaks at Evf2 positively (+) and negatively (-) regulated Dlx5/6UCEins (4Cseq, previously reported in ref. 5) across chr6. Two-tailed Student’s t test, significant pvals in red: (-) SMARCC2 120 bp, p = 4.1E-16, (-) SMARCC2 150 bp, p = 2.3E-08, (+)SMARCC2 120 bp, p = 5.7E-06, ( + ) SMARCC2 150 bp, p = 5.0E-07, ( + ) NONO 120 bp, p = 0.0009), blue (Evf2TS/TS) yellow (Evf2+/+). (+) SMARCC2 150 bp [Evf2TS/TS]: min = 0, max=2.13, median =1.92, Q1 = 1.87, Q3 = 1.97, lower boundary= 1.72, upper boundary= 2.13, (+) SMARCC2 150 bp [Evf2+/+]: min = 1.68, max=5.22, median =2.20, Q1 = 1.98, Q3 = 2.57, lower boundary= 1.09, upper boundary= 3.47). (-) SMARCC2 150 bp [Evf2TS/TS]: min=1.36, max=2.13, median = 1.92, Q1 = 1.87, Q3 = 1.97, lower boundary= 1.73, upper boundary= 2.11, (-) SMARCC2 150 bp [Evf2+/+]: min = 1.69, max = 4.35, median = 2.08, Q1 = 1.94, Q3 = 2.52, lower boundary= 1.06, upper boundary= 3.40. (+) SMARCC2 120 bp [Evf2TS/TS]: min=0, max=4.02, median =0, Q1 = 0, Q3 = 3.57, lower boundary = -5.36, upper boundary=8.93, (+) SMARCC2 120 bp [Evf2+/+]: min=0, max=8.37, median =3.69, Q1 = 0, Q3 = 4.06, lower boundary = −6.08, upper boundary = 10.14. (-) SMARCC2 120 bp [Evf2TS/TS]: min=0, max=4.00, median=0, Q1 = 0, Q3 = 0, lower boundary=0, upper boundary=0, (-) SMARCC2 120 bp [Evf2+/+]: min=0, max=6.01, median =3.56, Q1 = 2.37, Q3 = 3.98, lower boundary = -0.06, upper boundary= 6.41. (+) NONO 150 bp [Evf2TS/TS]: min=0, max=2.22, median =1.88, Q1 = 1.81, Q3 = 1.94, lower boundary= 1.60, upper boundary= 2.15, (+) NONO 150 bp [Evf2+/+]: min=1.74, max=2.10, median=1.94, Q1 = 1.89, Q3 = 1.98, lower boundary=1.74, upper boundary= 2.13. (-) NONO 150 bp [Evf2TS/TS]: min=1.67, max = 2.43, median =1.90, Q1 = 1.85, Q3 = 1.99, lower boundary=1.65, upper boundary = 2.19, (-) NONO 150 bp [Evf2+/+]: min=1.74, max=2.29, median=1.93, Q1 = 1.86, Q3 = 1.99, lower boundary=1.67, upper boundary=2.18. (+) NONO 120 bp [Evf2TS/TS]: min=0, max=4.34, median =0, Q1 = 0, Q3 = 0, lower boundary=0, upper boundary=0, (+) NONO 120 bp [Evf2+/+]: min=0, max=4.03, median =0, Q1 = 0, Q3 = 3.89, lower boundary = −5.83, upper boundary = 9.72. (-) NONO 120 bp [Evf2TS/TS]: min=0, max=4.99, median =0, Q1 = 0, Q3 = 0, lower boundary= 0, upper boundary=0, (-) NONO 120 bp [Evf2+/+]: min=0, max=4.37, median =0, Q1 = 0, Q3 = 3.88, lower boundary = −5.82, upper boundary= 9.70.

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