Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs

Abstract

Eukaryotic transcriptomes are characterized by widespread transcription of noncoding and antisense RNAs, which is linked to key chromosomal processes, such as chromatin remodelling, gene regulation and heterochromatin assembly. However, these transcripts can be deleterious, and their accumulation is suppressed by several mechanisms including degradation by the nuclear exosome. The mechanisms by which cells differentiate coding RNAs from transcripts targeted for degradation are not clear. Here we show that the variant histone H2A.Z, which is loaded preferentially at the 5' ends of genes by the Swr1 complex containing a JmjC domain protein, mediates suppression of antisense transcripts in the fission yeast Schizosaccharomyces pombe genome. H2A.Z is partially redundant in this regard with the Clr4 (known as SUV39H in mammals)-containing heterochromatin silencing complex that is also distributed at euchromatic loci, and with RNA interference component Argonaute (Ago1). Loss of Clr4 or Ago1 alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that as well as performing other functions, H2A.Z is a component of a genome indexing mechanism that cooperates with heterochromatin and RNAi factors to suppress read-through antisense transcripts.

Figure 1. Δpht1 causes upregulated antisense transcripts

a–b, H2A.Z is targeted preferentially to the 5' ends of genes. a, ChIP analysis of Swr1 and Msc1. DNA isolated from ChIP and whole cell extract (WCE) was analyzed by multiplex PCR. Control corresponds to gene-free region adjacent to SPAC5H10.03. Relative enrichment values are shown. b, H2A.Z deposition requires Swr1 and Msc1. c, H2A.Z is slightly enriched at heterochromatin domains. H3K4me, H3K9me and H2A.Z distributions at a subtelomeric region. Note the LTR cluster demarcating the border of the H2A.Z depleted region. d, H2A.Z is enriched at 5' ends of genes. e–g, Upregulation of antisense transcripts at convergent genes in Δpht1 cells. e, Percentage of genes with >2-fold altered median transcript levels in Δpht1. f, Strand-specific RT-PCR of RNAs isolated from wt and Δpht1 cells. g, Transcripts at rps2402/csx2 locus were examined by expression analysis using tiling microarray.

Figure 2. H2A.Z acts in synergistic manner with ClrC and Ago1 to suppress antisense transcripts

a, Cumulative derepression of antisense RNAs estimated as a percentage of genes with upregulated antisense. b, Representative examples with upregulated antisense in Δpht1Δclr4 and Δpht1Δago1 mutants. Signal intensity of transcripts from reverse/lower strand is plotted. c, Profile of average antisense signal ratios at convergent genes in single (Δpht1/wt, Δago1/wt and Δclr4/wt) and double mutants (Δpht1Δclr4/wt and Δpht1Δago1/wt). Double alignment at 5' and 3' ends of genes was applied and varied gene length compensated by compression of middle part of gene. H2A.Z localization is indicated in green. d, Cumulative profile of antisense transcripts in indicated strains. The average antisense signal profile was calculated for genes with increased levels of antisense in double mutants and compared with the profile in wt.

Figure 3. Read-through antisense transcripts are suppressed by the exosome

a, Antisense transcripts upregulated in Δpht1Δclr4 cells result from overlapping transcription at convergent genes. Northern analyses of RNAs at SPBC16h5.04-cyp7. Probes complementary to antisense (probe #1 and #2) and sense portion (probe #3) of SPBC16h5.04 detected read-through transcripts. Oligonucleotide-targeted RNase H cleavage was used to determine 3' end of transcript. b, Cumulative profile of antisense transcripts in Δrrp6 and wt cells. The antisense profile was calculated for genes with up-regulated antisense in Δrrp6 cells and compared with wt. c, Expression profiling of antisense transcripts in Δpht1Δclr4 and Δrrp6 cells.

Figure 4. H2A.Z and heterochromatin factors suppress antisense RNAs targeted by the exosome

a, Density plot comparing upregulation of antisense transcripts in indicated mutants. Median antisense upregulation (mutant/wt) was calculated for 842 genes. Pearson's correlation coefficient (r) and the p value of the linear regression are indicated. Panel comparing antisense profiles of two rrp6 mutants illustrates little variation between biological replicates. b, Hierarchical clustering of mutants based on similarities of their antisense profiles. Pairwise comparisons of antisense profiles were performed as in Fig. 4a and Pearson's correlation coefficients were converted into color codes. c, Model for antisense suppression at convergent genes. H2A.Z at the 5' ends of genes contribute to suppression of read-through transcripts that are degraded by exosome. Antisense suppression also requires ClrC and Ago1, which along with H2A.Z may facilitate loading of other factors to block Pol II progression and/or mediate the processing of RNAs by the exosome.