Figure 4. EBdCas9 reveals functional distal TATA box.

(A) TATA box and initiator (INR) representation with respect to g6 locus using the Element Navigation Tool for detection of core promoter elements.

(B) Generation of TATA box deletion (TATA ∆) clones TATA ∆ #1 and TATA ∆ #2 using CRISPR-Cas9. gRNA1 and −2 excise an 88 bp fragment, including the g6 region. The g6 region was reconstructed using a 168 bp single-stranded DNA (ssDNA) donor. Depicted here is a deletion of 40 bp including the TATA box and 3 initiator elements (INR). Genomic DNA of TATA ∆ #1 or TATA ∆ #2 displaying homozygous clones of 40 bp TATA box deletion using Sanger sequencing. PAM-site-blocking mutation is denoted with a red asterisk.

(C) qRT-PCR of EBdCas9 and TBX18 relative fold change after 3 days of Dox induction and TBX18 g6 RNA transfection normalized to β-actin and compared with no guide for each respective cell line (WT: EBdCas9, TATA ∆ #1, TATA ∆ #2).

(D) EBdCas9 WT and EBdCas9 TATA ∆ #1 and TATA ∆ #2 timeline for Dox induction, gRNA transfection, and ChIP-qPCR analysis using the antibodies Cas9, EZH2, TATA binding protein (TBP), and RNA Pol II and analyzing for TBX18 g6 DNA region. Right panel depicts 4 possible model scenarios for TATA box and TATA ∆ with and without EBdCas9.

(E) ChIP-qPCR of induced (+Dox) EBdCas9 (WT) and EBdCas9 TATA ∆ #1 and TATA ∆ #2 3 days following transfection with TBX18 g6 RNA (+g6) or no transfection (–g). Normalized to fraction input/H3. Antibodies that were used for ChIP are listed above the graphs, and the genomic region analyzed by qPCR includes the TBX18 g6 locus (n = 3, SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test).

(F) Model describing EBdCas9/TBX18 mode of operation: (I) The TBX18 distal TATA box function is repressed by PRC2. (II) EBdCas9 derepresses the TBX18 TATA box region. (III) TBP, TBP associated factors (TAFs), and RNA Pol II are recruited to the TBX18 TATA box region to allow TBX18 transcription.