Fig. 3: Silencing and promoter targeting is limited to a 200 nucleotide mOct4P4 lncRNA region.

a Schematic representation of generated mOct4P4-24xMS2 deletion constructs. Gray boxes, sequences with homology to Oct4/OCT4 5′UTR; gray lines, sequences with homology to Oct4/OCT4 3′UTR. 334 bp-spliced sequences are present in mOct4P4 and −200mOct4P4 sequences; deleted regions are indicated. White boxes indicate identified 200 nucleotide mOct4P4 region. 24xMS2 RNA stem loop motifs at the 3′ end of mOct4P4 deletion constructs are indicated. Arrows indicate the locations of RT-PCR primers. b qRT-PCR determining 200 bp-mOct4P4 and −200 bp-mOct4P4 expression levels in experimental mESCs. Expression values were normalized to Gapdh. c Subcellular localization of lncRNAs derived from constructs in (a). Expression values are shown as percentage of total RNA levels, as determined by qRT-PCR. d, e qRT-PCR (d) and western blot analysis (e) using mESCs ectopically expressing mOct4P4, 200 bp-mOct4P4, and −200 bp-mOct4P4 constructs. Oct4 expression values were normalized against Gapdh (d) or ACTIN (e). Shown numbers represent OCT4/ACTIN ratio as mean of three independent experiments (control was set “100”) (e). f, g ChIP analysis of Oct4 promoter region in mESCs stably overexpressing indicated constructs and using described antibodies. qRT-PCR was performed to measure promoter enrichment. Only mOct4P4 and 200 bp-mOct4P4 constructs localize to the Oct4 promoter (f) and drive H3K9me3 enrichment (g). Error bars represent standard deviation. Precise p values are indicated. n: number of independent experiments carried out.