Excision of H3K4me3/DNaseI signatures abolishes ncRNA expression.

A) Experimental overview. U937 monocytes are spin-lipofected with dual guideRNA CRISPR constructs and clonally expanded after single cell sorting of cells positive for GFP (co-expressed from the CRISPR vector). B) Genomic PCR showing homozygous deletion of the proximal promoter elements identified in Fig 2 of the MALAT1, miR155, miR146a genes after clonal expansion of monocytes transfected with the respective dual guideRNA CRISPR constructs (KO). Transfection of control CRISPR construct does not result in the deletion, respectively (WT). C) Real-time PCR analysis of IL1β mRNA expression in wild type (WT) and MALAT1, miR-146a or miR-155 TSS knockout monocytes after control-stimulation (mock) or activation with 1 μg / ml of LPS for 4h, 8h or 16h. Fold-changes compared to mock-stimulation of control cells are shown. D) Real-time PCR analysis of MALAT-1 expression in wild-type (WT) or in MALAT1 H3K4me3/DNaseI element deficient cell clones stimulated as in C). E) Northern blot with miR-146a, and miR-17 detection probes showing loss of miR-146a induction on stimulation with LPS (1 μg / ml) in monocytes deficient in the gene-proximal H3K4me3/DNaseI element (KO) but not in wild-type (WT) cell clones. F) Same as E) but with miR-155 gene proximal H3K4me3/DNaseI element excised and miR-155 instead of miR-146a detection.