A Large Fraction of Extragenic RNA Pol II Transcription Sites Overlap Enhancers (original) (raw)
Figure 3
Characterization of the extragenic transcripts generated upstream of LPS-inducible genes.
(A) Polyadenylation of extragenic Ccl5 transcripts. Total RNA was reverse-transcribed using oligo-dT primers. cDNA was then amplified with primers corresponding to regions −1, −2, and −3 upstream of Ccl5 (as in Figure 1). (B) Upstream extragenic transcripts are nuclear RNAs. Macrophages were fractionated before RNA extraction. RNA from the cytoplasmic and nuclear fractions was then reverse transcribed and amplified with the indicated primers. Neat1 is a nuclear non-coding RNA that was used as a control of the fractionation procedure. (C) Extragenic transcription upstream of Ccl5 generates long unspliced transcripts. RNA was reverse transcribed using antisense primers in the region just upstream of Ccl5 TSS, as indicated. cDNA was then PCR-amplified using primers in the extragenic region −1 (as in Figure 1A). (D) Extragenic Ccl5 and Cxcl11 transcripts are very unstable. Cells were stimulated with LPS for 2 h, followed by a 30 min actinomycinD (5 µg/ml) chase. mRNAs for Ccl5 and Cxcl11 and the corresponding extragenic transcripts were measured by quantitative PCR. UT, untreated. (E) DRB insensitivity of extragenic Ccl5 and Cxcl11 transcripts. Macrophages were stimulated with LPS for 2 h in the presence or absence of DRB (50 µg/ml), as indicated. UT, untreated.