brd2_ppt.pdf

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1. Experimental validation of long-range RNA interactions in human Brd2&Brd3 genes 1 Petrova Marina 06 Sep. 2023
2. BET family proteins 2 • bromodomain protein • recognize acetylated lysine & recruits transcription factors • small molecules in targeted cancer therapy a remarkable conservation of exon boundari Figure S1), and the phylogenetic trees derive to BRD3, not BRD4 (Figure S2). BRD3 mm BRD3 hs BRD2 mm BRD2 hs BRD4 mm BRD4 hs BRDT mm BRDT hs fs(1)h_dm 0.91 1.09 0.67 1.28 0.27 0.52 0.2 0.12 0.14 0 0 Tree scale: 1 0.05 0.03 0.02 0.05 0.06 0.04 0.04 (A) PB1-4 PB1-2 PB1-5 SM ARCA2A SMARCA4 PB1-3 PB1-1 ASH1L PB1-6 SMARCA2B TAF1L-2 TAF1-2 TAF1L-1 TAF1-1 ZMYND8 ZM YND11 PHI P-1 BRW D3-1 BRW D1-1 BRD9 BRD1 BRPF1B BRPF3 ATAD2 ATAD2B BRPF1A BRD7 BRD3-2 BRD2-2 BRD4-2 BRDT-2 BRD3-1 BRD2-1 BRD4-1 BRDT-1 BRD8-2 BRD8-1 EP300 CREBBP BRW D1-2 BRW D3-2 BAZ1B PHI P-2 MLL KIAA2026 BAZ2B BAZ2A TRIM33A TRIM24 TRIM66 TRIM28 SP140L SP140 SP100 SP110A TRIM33B SP110B KAT2A BPTF CECR2 BAZ1A KAT2B 1 2 3 4 5 6 7 8 BAZ2B B BAZ2A A TRIM33A TRIM24 TRIM66 TRIM28 SP140L L SP140 SP100 SP110A TRIM33B SP110B 7 MLL KIAA2026 8 KAT2A BPTF CECR2 BAZ1A KAT2B 1 BRD8-2 BRD8-1 EP300 CREBBP BRW D1-2 R R BRW D3-2 R R BAZ1B PHI P HI HI -2 P P 2 BRD3-2 BRD2-2 BRD4-2 BRDT-2 BRD3-1 BRD2-1 BRD4-1 BRDT-1 3 TAF1L-2 TAF1-2 T T TAF1L-1 T T TAF1-1 ZMYND8 ZM YND11 PHI P HI HI -1 P P BRW D3-1 BRW D1-1 4 2 PB1-4 PB1-2 PB1-5 SM ARCA2A SMARCA4 PB1-3 PB1-1 ASH1L L PB1-6 6 SMARCA2B 5 BRD9 BRD1 BRPF1B BRPF3 ATAD2 ATAD2B BRPF1A A BRD7 6 B A B (A)
3. Brd2&Brd3 RNA structures 3 (A) chr6: 32,974,500 32,975,000 32,975,500 Gene Annotation Set from GENCODE v. 33 100 vertebrates conservation by PhastCons Pfam Domains in GENCODE Genes BRD2 BRD2 ΔG=-29.1 kcal/mol Bromodomain PCCR ΔG <= -25 kcal/mol R1 R2 3 3b 4 (C) Figure 1: The BET fam (B) chr9: 134,048,500 134,049,000 134,049,500 134,050,000 134,050,500 Gene Annotation Set from GENCODE v. 33 GTEx novel transcripts PCCR ΔG <= -25 kcal/mol 100 vertebrates conservation by PhastCons Pfam Domains in GENCODE Genes BRD3 Bromodomain
   G=-34.3 kcal/mol BRD3 R3 R4 5 5b 6 (D) Brd2 Brd3
4. Brd2 Brd3 Brd4 exons 4 55 %5'7 %URPRGRPDLQ %URPRGRPDLQ E E E %5' %5' %5' (B)
5. ASO Treatment of A549# 48h 5 • brd2 2 3 2 2a 3 171bp 79bp * * 0.00 0.25 0.50 0.75 1.00 con clna 5nM 25nM 100nM psi ASO1 0.00 0.25 0.50 0.75 1.00 con clna 5nM 25nM 100nM psi ASO2
6. Minigene mutagenesis 6 GC UGA UGCGGCCCC GGGG CC CG CGA GGGG CC CG GCUGCGGCCCC CGA GGGG CC CG ACUGCGGCCCC CGA GGGG CC CG ACUGCGGCCCC wt m1 m2 m1m2
7. Minigene mutagenesis Brd2 7 2 3 2 2a 3 171bp 79bp ** ** 0.00 0.25 0.50 0.75 1.00 wt m1 m2 m1m2 psi
8. ASO treatment of A549# (Brd2) 48h 8 0.5 1.0 1.5 2.0 2.5 con clna 5nM 25nM 100nM fold_change brd2_expression ASO1 0.50 0.75 1.00 1.25 1.50 con clna 5nM 25nM 100nM group fold_change brd2_expression ASO2
9. Brd3 ASO treatment of A549# 9 alt 0.01 0.02 0.03 psi ASO1 brd3 0 C 5 25 100 200 100 150 300 75 50 25 brd3 216+134bp 0 C 5 25 100 ** **
10. Minigene mutagenesis Brd3 10 alt 200 100 150 300 75 50 25 brd3 216+134bp wt m1 m2 m1m2 MQ 0
11. Rbfox2 overexpression in 3 cell lines 11 0.15 0.20 0.25 0.30 0.35 con oe psi Rbfox2_OE brd2 con oe con oe 22Rv1 LNCaP PC-3 • oe – Doxycycline-inducible overexpression of Rbfox2 • con – no doxycycline added
12. Brd3 cryptic exon inclusion 12 200 100 150 300 700 400 500 75 50 25 brd3 216+134bp U-87 U-251 22Rv1 22Rv1 LNCaP LNCaP PC-3 PC-3 RBFOX2_OE RBFOX2_OE RBFOX2_OE alt • Several cell cultures and Rbfox2 overexpression in 22Rv1, LNCaP, PC-3
13. Brd3 cryptic exon inclusion 13 200 100 150 300 700 400 500 75 50 25 A549 HEK HeLa IMR32 MCF7 A549+CHX HepG2 Skbr3 Sk-N-MC brd3 ???+134bp alt
14. ASO treatment of A549# 24h (Brd2) 14 0.1 0.2 0.3 0.4 0.5 psi ASO2 brd2_psi con clna 5nM 100nM con clna 5nM 25nM 100nM + CHX – CHX 0.2 0.3 0.4 0.5 con clna 5nM 100nM con clna 5nM 25nM 100nM psi ASO1 brd2_psi + CHX – CHX
15. ASO treatment of A549# 24h (Brd2) 15 0.8 1.0 1.2 1.4 fold_change ASO2 brd2_expression con clna 5nM 100nM con clna 5nM 25nM 100nM + CHX – CHX 0.6 0.8 1.0 1.2 fold_change ASO1 brd2_expression con clna 5nM 100nM con clna 5nM 25nM 100nM + CHX – CHX *
16. ASO and CHX treatment of A549# 48h (Brd2) 16 0.15 0.20 0.25 0.30 0.35 0.40 psi ASO2 brd2_psi con 100nM con 5nM 100nM + CHX – CHX 0.2 0.3 0.4 0.5 psi ASO1 brd2_psi con 5nM 100nM con 5nM 100nM + CHX – CHX
17. ASO and CHX treatment of A549# 48h (Brd2) 17 0.50 0.75 1.00 1.25 1.50 con 5nM 100nM fold_change ASO1 brd2_expression con 5nM 100nM + CHX – CHX 0.9 1.0 1.1 1.2 1.3 fold_change ASO2 brd2_expression con 100nM con 5nM 100nM + CHX – CHX
18. ASO treatment of A549# Brd2_minigene 18 0.00 0.25 0.50 0.75 1.00 con lna1_25 lna1_50 lna2_25 lna2_50 fold_change expression brd2 minigene + ASO1/2 0.4 0.5 0.6 0.7 0.8 con lna1_25 lna1_50 lna2_25 lna2_50 psi brd2 minigene + ASO1/2
19. Brd2 putative regulators 19 • SF3B1 • Rbfox2
20. Sf3b4, Sf3b1 siRNA treatment of A549# 20 0.0 0.5 1.0 con luc sirna1_10 sirna1_100 sirna2_10 treatment ddct sf3b1 0.00 0.25 0.50 0.75 1.00 1.25 con luc sirna_10 sirna_100 treatment ddct sf3b4
21. Sf3b4, Sf3b1 siRNA treatment of A549# 21 0.0 0.1 0.2 0.3 con luc sf3b1_sirna_10 sf3b4_sirna_10 sf3b4_sirna_100 treatment psi brd2 Inclusion_percent 0.0 0.1 0.2 0.3 0.4 0.5 con luc sf3b1_sirna_100 sf3b4_sirna_100 treatment psi brd2 Inclusion_percent +CHX
22. Sf3b1 siRNA treatment of HepG2# 22 0.15 0.20 0.25 0.30 0.35 0.40 luc psi SF3B1_KD brd2 KD luc KD +CHX - CHX