Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5

Abstract

Long non-coding RNAs (lncRNAs) have been shown to epigenetically regulate certain genes in human cells. Here we report evidence for the involvement of an antisense lncRNA in the transcriptional regulation of the pluripotency-associated factor Oct4. When an lncRNA antisense to Oct4-pseudogene 5 was suppressed, transcription of Oct4 and Oct4 pseudogenes 4 and 5 was observed to increase. This increase correlated with a loss of silent state epigenetic marks and the histone methyltransferase Ezh2 at the Oct4 promoter. We observed this lncRNA to interact with nucleolin and PURA, a 35 kD single-stranded DNA and RNA binding protein, and found that these proteins may act to negatively regulate this antisense transcript.

Figure 1

(A) Schematic representation of transcripts, primer sites, siRNA target sites and other genetic elements associated with Oct4 and Oct4-pg5. (B) PCR of MCF-7 RNA depleted of polyadenylated transcripts and converted to cDNA in a strand-specific manner using indicated primers. PCR products were analyzed on 6% polyacrylamide gel. Non-polyadenylated antisense transcripts were detected overlapping the coding region of Oct4-pg5 and the coding and promoter regions of Oct4.

Figure 2

(A) Screen of three siRNAs targeted to lncRNAs antisense to Oct4 and Oct4-pg5. Compared to cells treated with the control siRNA, samples transfected with siO18 and siO98 demonstrated a decrease in antisense and an increase in sense transcripts associated with Oct4 and/or Oct4-pg5. (B) Forty-eight hours post-transfection with shO18, relative to samples treated with the scrambled control shS18, antisense transcripts associated with Oct4 and/or Oct4-pg5 were decreased, while sense transcripts were not significantly changed. (C) Seventy-two hours post transfection with shO18, antisense transcripts associated with Oct4 and/or Oct4-pg5 were decreased, while sense transcript levels were significantly increased. (D) Seventy-two hours post-transfection with shO18, antisense transcripts associated with Oct4 were unchanged, while sense transcript levels were significantly increased. (B–D) Averages are shown from cultures treated in triplicate, error bars represent standard errors of means and p values from two-sided t-tests are indicated.

Figure 3

(A–C) Compared to cells treated with the scrambled control, samples transfected with shO18 demonstrated a significant increase in transcription of Oct4-pg5, Oct4 and Oct4-pg4. (D) Transfection of MCF-7 cells with siRNAs targeted to Ezh2 and G9a resulted in an increase in Oct4 sense transcript levels. (E) Treatment of MCF-7 cells with shO18 resulted in a loss of the silent state epigenetic marks H3K9me2 and H3K27me3 and the histone methyltransferase Ezh2 at the Oct4 promoter. (A–D) Values obtained from qPCR were standardized to β-actin levels. (A–E) Averages are shown from cultures treated in triplicate, error bars represent standard errors of means and p values from two-sided t-tests are indicated. Samples were analyzed 72 h post-transfection.

Figure 4

(A) Schematic of biotin-mediated pulldown of target asRNA. (B) RNA samples isolated from nuclei incubated with biotin-tagged O18 or control (miRN367) oligonucleotides were reverse transcribed with Oct4 Set 1 Fwd, amplified by PCR using Oct4 set 1 and analyzed on a 6% polyacrylamide gel. (C) Isolated proteins were analyzed by mass spectrometry and the indicated proteins, unique to samples pulled-down with biotin-tagged O18, were identified.

Figure 5

(A) Transfection of MCF-7 cells with shRNAs targeted to PURA and NCL resulted in a decrease in Oct4 sense transcript levels. Results have been normalized to internal β-actin levels. (B) Treatment of MCF-7 cells with a shRNA targeted to PURA resulted in an enrichment of the silent state epigenetic mark H3K27me3 at the Oct4 promoter. (C) Transfection of MCF-7 cells with shRNAs targeted to indicated genes resulted in no change in asOct4 or asOct4-pg5 levels. (A–C) Averages are shown from cultures treated in triplicate, error bars represent standard errors of means and p values from two-sided t-tests are indicated. Samples were analyzed 72 h post-transfection.

Figure 6

(A) The non-coding transcript asOct4-pg5 associates with Ezh2, G9a and possibly additional regulatory proteins. This complex localizes to the Oct4 promoter where it induces heterochromatization and inhibition of transcription. PURA and NCL may also bind asOct4-pg5, but function to inhibit its activity by sequestration. (B) When PURA and NCL are suppressed by RNAi, asOct4-pg5 is able to target Ezh2 and G9a to the Oct4 promoter, resulting in reduced Oct4 transcription.