TALE Factors Recruit RNA Polymerase II but Drive Only Weak hoxb1a Transcription

(A–I) In situ hybridization to hoxb1a transcripts in 4 hpf zebrafish embryos injected with mRNA and MOs as indicated in lower right corner of each panel. Arrows indicate examples of positive signals.

(J and K) Quantitative PCR detection of hoxb1a transcripts in 4 hpf zebrafish embryos injected with the indicated mRNAs and MOs.

(L) Occupancy of serine-5 phosphorylated RNA polymerase II at the hoxb1a promoter of 4 hpf embryos overexpressing TALE factors or TALE factors + Hoxb1b.

Data are presented as the average of a minimum of three repeats (except in K, which was done in duplicate) with error bars indicating SD. Statistical significance was determined using Student’s t test in Microsoft Excel; p values < 0.05 are indicated.

Hoxb1b triggers P-TEFb-Mediated Activation of RNA Polymerase II

(A–F) Embryos were injected with mRNA as indicated in lower right corner of each panel and incubated in DMSO (A and B), 10 µM flavopiridol (C and D), or 35 µM flavopiridol (E and F) until 4 hpf, followed by in situ hybridization to detect hoxb1a expression. Arrows point to examples of hoxb1a expression.

(G) Quantitative PCR detection of hoxb1a transcripts in 4 hpf zebrafish embryos injected with the indicated mRNAs and treated with the indicated concentrations of flavopiridol.

(H) CDK9 (the catalytic component of P-TEFb) occupancy at a region 10 kb upstream of the hoxb1a promoter, at the TALE:Hox binding site (MPH), at the transcription start site (TSS), or at Exon 2 in 4 hpf embryos overexpressing TALE factors or TALE factors + Hoxb1b.

(I) Quantitative PCR detection of HoxB1 transcripts in F9 cells transfected with the indicated plasmids.

(J) Western blot detection of transcription factors following transfection of F9 cells.

(K) Quantitative PCR detection of HoxB1 transcripts in F9 cells transfected with the indicated plasmids and treated with the indicated concentrations of flavopiridol.

(L) CDK9 occupancy at the TALE:Hox binding site (MPH) and the transcription start site (TSS) of the HoxB1 gene in F9 cells transfected with the indicated plasmids.

(M) Occupancy of serine-2 phosphorylated RNA polymerase II at the hoxb1a transcription start site (left) or exon 2 (right) of 4 hpf embryos overexpressing TALE factors or TALE factors + Hoxb1b.

(N) Ratio of Prep1+Pbx4+Hoxb1b signal/Prep1+Pbx4 signal for various parameters related to hoxb1a expression.

Data are presented as the average of a minimum of three repeats with error bars indicating SD. Statistical significance was determined using Student’s t test in Microsoft Excel; p values < 0.05 are indicated.