Fig. 2: SMANTIS interacts with RUNX1.

a Genomic structure and LentiCRISPRv2-mediated knockout strategy of SMANTIS. The combination of gRNA1 and gRNA2 was used for CRISPR/Cas9 of SMANTIS in THP-1 cells. gRNA, guide RNA. b Agarose gel electrophoresis after PCR of the genomic DNA region of SMANTIS. NTC, non-targeting control gRNA. GAPDH serves as loading control. c SMANTIS expression with and without CRISPR/Cas9 mediated KO as determined by RT-qPCR. n = 4, Mann-Whitney test. d RNA in situ hybridization of THP-1 cells with (KO) and without (NTC) CRISPR/Cas9 mediated KO of SMANTIS with RNAscope. Arrows indicate prominent SMANTIS expression. Representative images are shown. Scale bar indicates 10 µm. e IGV genome browser view of SMANTIS transcription start site (TSS) with tracks for RNA-Seq and Assay for Transposase-Accessible Chromatin (ATAC) of THP-1 cells with SMANTIS KO compared to control (NTC). The ATAC-seq signal in the KO is strongly reduced indicating successful gRNA-mediated deletion (scissors). f Volcano plot of RNA antisense-pulldown with oligonucleotides targeting SMANTIS in NTC versus SMANTIS KO THP-1. Red dots indicate protein interactions of SMANTIS, which are lost after SMANTIS KO. The negative log-transformed p values are shown on the y-axis. n = 4, students t-test. g RNA-immunoprecipitation (RIP) with antibodies against RUNX1 or IgG followed by RT-qPCR of SMANTIS. n = 5, paired t-test. h, i Scheme of SMANTIS mutants used (h) for the in vitro binding assay with recombinant RUNX1 followed by RT-qPCR (i). n = 5, One-way ANOVA with Dunnett’s post hoc test. Numbers indicate exon number; FL, full length; A, Alu element; N, N-terminus; C, C-terminus. j, k Scheme of RUNX1 mutants (j) used for the in vitro binding assay with the Alu element of SMANTIS followed by RT-qPCR (k). n = 3. One-way ANOVA with Dunnett’s post test. RUNT, DNA-binding domain; TAD, transcription activation domain. Error bars are mean +/− SD. *p < 0.05. KO, knockout.