Figure 2: Overlapping lncRNA transcription localizes H3K36me3 and Rpd3S to target promoters.

(a) AAD10 has a constitutively active distal promoter (blue arrow) and a proximal promoter (red arrow) induced during galactose incubation in set2Δ. SUL1 has a proximal promoter (red arrow) activated in galactose media in set2Δ and a constitutively active antisense promoter (blue arrow). Light blue boxes show open reading frame (ORF) positions and orange box shows lncRNA transcription. Time course as in Fig. 1a is arrayed from bottom to top for the indicated strains. Increased blue colour indicates more transcript hybridizing to the array. (b) Histone methylation patterns of AAD10 and SUL1 from cells grown in YPD were analysed using the data set from Pokholok et al.¹¹. Whereas the AAD10 distal promoter and SUL1 antisense promoter have high levels of H3K4me3, the core mRNA promoters have high levels of H3K36me3. White boxes show the ORF position. (c) Northern blot analysis of AAD10 transcript with a 3′-strand-specific DNA probe. The indicated cells were grown in synthetic complete (SC) medium containing raffinose (Raf) and shifted to SC-galactose media for 120 min (Gal120). Bottom panels show two transcripts of AAD10 detected by northern blot analysis, which are schematicized at top. Blue arrow is a distal promoter that produces a lncRNA and red arrow is a proximal promoter for AAD10 mRNA transcription. A bar underneath upper panel indicates position of probe used for northern blot analysis. (d) The Set2–Rpd3S pathway deacetylates histones at the AAD10 core promoter. Crosslinked chromatin from the indicated strains grown in YPD (where set2Δ also derepresses AAD10) was precipitated with anti-H3 or anti-acetyl H4 as indicated. PCR analysis of the precipitated DNA was carried out on both distal (blue bars) and proximal (red bars) promoters of AAD10. A non-transcribed region near the telomere of chromosome VI was used for an internal control. The signals for acetyl H4 were quantitated and normalized to the total H3 signal, and the ratios were graphed. Error bars show the s.d. calculated from two biological replicates, each with three technical replicates. Similar results were obtained with cells grown in galactose for 120 min. WT, wild type.