Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF

Abstract

Approximately 75% of the human genome is transcribed, the majority of which does not encode protein. However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from ∼57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF's function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.

Keywords: chromatin remodeling; esBAF; ncRNA; nucleosome occupancy; transcription.

Figure 1.

The esBAF complex inhibits production of noncoding transcripts initiating from multiple genomic features. Genome browser tracks of two DHSs (A,B), two TSSs (C,D), two intragenic regions (E,F), and two TTSs (G,H). Bars corresponding to normalized CapSeq reads from EGFP knockdown (KD) and Smarca4 knockdown are shown in log₂ scale at their initiation sites, and the (real space) number of normalized reads per cluster of transcripts is noted in each track. Browser tracks of normalized CapSeq reads of one replicate from EGFP knockdown and Smarca4 knockdown are shown. Isoforms are shown in an orange box below the scale, with introns indicated as black lines. DHSs are indicated in green boxes. Blue bars indicate transcription from the Crick strand, while red bars indicate transcription from the Watson strand. It should be noted that transcripts near TSSs in the same orientation as the coding sequence likely depict mRNAs not ncRNAs.

Figure 2.

Up-regulation of ncRNAs from gene-distal and gene-proximal regions upon esBAF depletion. (A–C) Histogram of normalized sense and antisense transcripts obtained from RNA-seq analysis surrounding DHSs (A), TSSs (B), and TTSs (C) in EGFP knockdown (KD) and Smarca4 knockdown ESCs. (D) Distribution of ncRNAs significantly misregulated upon Smarca4 knockdown in ESCs. (E–H) Heat maps quantifying noncoding transcripts surrounding DHSs (±500 bp) (E), TSSs (−500 to +100 bp) (F), intragenic regions (antisense only, >500 bp from the TSS) (G), or TTSs (antisense only, −500 to +500 bp from the TTS) (H) in averaged biological replicates of EGFP knockdown and Smarca4 knockdown ESC RNA-seq experiments. Expression is indicated as log₂(normalized reads). (I) RT-qPCR validation of ncRNA transcripts initiating from gene-distal DHSs. Expression levels (mean ± SD values of three biological replicates) upon Smarca4 knockdown are shown relative to the EGFP knockdown. (J) Increased antisense transcript production from 14 coding gene promoters upon knockdown of multiple esBAF subunits. Analyses were performed as in I. (*) P < 0.05; (**) P < 0.01; (ns) not significant.

Figure 3.

esBAF promotes nucleosome depletion over DHSs and elevated nucleosome occupancy flanking DHSs in ESCs. (A) Heat maps of nucleosome occupancy obtained by MNase-seq over gene-distal DHSs ±2 kb in EGFP knockdown (KD) (left) and Smarca4 knockdown (right) ESCs. Nucleosome occupancy is indicated in yellow. DHSs were called from GSM1014154 with TSSs removed, since they are considered separately (see below). (B,C) Aggregation plot of relative nucleosome occupancy upon EGFP knockdown or Smarca4 knockdown over all gene-distal DHSs (B) or over gene-distal DHSs that are either highly or lowly bound by esBAF (C). The P-value for the change in flanking nucleosome is indicated. (D) Validation of changes in occupancy of nucleosomes immediately flanking DHSs. Histone H3 levels of nucleosomes flanking seven DHSs found to exhibit reduced occupancy in the Smarac4 knockdown MNase-seq data set were determined in EGFP knockdown and Smarca4 knockdown ESCs by ChIP-qPCR along with four control DHS-proximal nucleosomes that were not altered upon Smarca4 knockdown. Histone H3 levels are expressed as a fraction of the input. Shown are the mean ± SD values of three biological replicates. (E) Validation of changes in nuclease accessibility over DHSs. Accessibility to MNase treatment was determined over seven DHSs altered in the Smarca4 knockdown MNase-seq data set in EGFP knockdown and Smarca4 knockdown ESCs by MNase-qPCR along with three control DHSs that were not altered upon Smarca4 knockdown. Protection relative to undigested chromatin is shown as the mean ± SD values of three biological replicates. For D and E, statistical significance is indicated by an asterisk. (*) P < 0.05; (**) P < 0.01; (ns) not significant.

Figure 4.

esBAF regulates NDRs and flanking nucleosome occupancy of promoters and TTSs. (A–C) Aggregation plot of relative nucleosome occupancy upon EGFP knockdown (KD) or Smarca4 knockdown over TSSs (A), TTSs (B), or Ctcf-binding sites (C) ±2 kb in ESCs. Ctcf-binding sites were based on published data (Chen et al. 2008). P-values indicating the statistical significance of changes in nucleosome occupancy are indicated. To calculate P-values, a standard t-test summing reads from −500 to −240 upstream of TSSs and +240 to +500 downstream from TTSs in EGFP knockdown and Smarca4 knockdown was performed. (D) Aggregation plot of relative nucleosome occupancy upon EGFP knockdown or Smarca4 knockdown over DHSs grouped by changes in ncRNA levels in Smarca4 knockdown ESCs. Displayed are the top 25% of DHSs with increased ncRNA levels upon Smarca4 knockdown and the bottom 25% of DHSs with decreased or no change in ncRNA levels upon Smarca4 knockdown. (E) Log₂ fold change of Smarca4 knockdown/EGFP knockdown ncRNA expression originating from annotated DHSs (left panel) or upstream of TSSs (right panel) from averaged ESC RNA-seq experiments, sorted by the log₂ fold change of Smarca4 knockdown/EGFP knockdown nucleosome occupancy adjacent to DHSs or upstream of TSSs (−1 position). (F,G) Receiver of operating characteristic (ROC) curves for changes in nucleosome occupancy relative to changes in expression for DHSs (F) and TSSs (G). The P-values indicating the significance of changes in flanking nucleosome occupancy curves relative to scrambled data are shown.

Figure 5.

esBAF silences ncRNA expression by increasing nucleosome occupancy adjacent to NDRs. (A) Diagram of the DHS-chr2 locus with qPCR amplicons depicted. (B) Histone H3 ChIP-qPCR over the −1 nucleosome in wild-type (WT) or nucleosome SB homozygote (SB/SB) lines upon either EGFP knockdown (KD) or Smarca4 knockdown. H3 levels are expressed as a fraction of the input. Shown are the mean ± SD values of three biological replicates. (C) Randomly primed RT-qPCR of DHS transcripts in EGFP knockdown or Smarca4 knockdown cells. Expression levels were normalized to GAPDH and are shown relative to wild-type EGFP knockdown. Shown are the mean ± SD values of three biological replicates. (D) Diagram of the Ttc25 locus with qPCR amplicons depicted. (E) Histone H3 ChIP-qPCR over the −1 nucleosome in wild-type, SB heterozygote (+/SB), or SB/SB lines upon either EGFP knockdown or Smarca4 knockdown. Data are depicted as in B. (F) Randomly primed RT-qPCR of antisense transcripts in EGFP knockdown (E) or Smarca4 knockdown (S) cells, as in C. Statistical significance of alterations in expression or occupancy is indicated. (*) P < 0.05; (**) P < 0.01; (ns) not significant.

Figure 6.

The nucleosome-mediated barrier to antisense ncRNA expression stimulates sense transcription at Ttc25 gene. (A) RNAPII or IgG control ChIP-qPCR upstream of the Ttc25 locus (primer sets indicated as in Fig. 5D) in wild-type (WT), +/SB, or SB/SB strains upon knockdown (KD) of either EGFP (E) or Smarca4 (S), expressed as a fraction of the input. Shown are the mean ± SD values of three biological replicates. (B) Random primed RT-qPCR of Ttc25 mRNA. Expression levels were normalized to GAPDH and are shown relative to wild-type EGFP knockdown. Shown are the mean ± SD values of three biological replicates. (C) RNAPII or IgG control ChIP-qPCR over the Ttc25 locus in wild-type, +/SB, or SB/SB strains upon either EGFP knockdown or Smarca4 knockdown for three biological replicates, displayed as in A. Statistical significance of alterations in expression or occupancy is indicated. (*) P < 0.05; (**) P < 0.01; (ns) not significant.

Figure 7.

Position-dependent effects of upstream nucleosomes on sense and antisense expression at the Ttc25 gene. (A–C) Diagram of the Ttc25 locus with alternative SB positions. The SB was moved 60 bp 5′ of the original −1 nucleosome (−60; A,D,G), 60 bp 3′ of the original −1 nucleosome (+60; B,E,H), or 180 bp 3′ of the original −1 nucleosome (+180; C,F,I). The location of the original −1 nucleosome is depicted as a transparent nucleosome. (D–F) Randomly primed RT-qPCR of antisense Ttc25 transcripts (as-Ttc25 RNA). Expression levels were normalized to GAPDH and are shown relative to wild-type (WT) EGFP knockdown (KD). Shown are the mean ± SD values of three biological replicates. (G–I) Randomly primed RT-qPCR of Ttc25 mRNA in alternative SB lines. (J) Model for esBAF regulation of ncRNAs. Upon loss of esBAF, nucleosome occupancy is altered, with increased occupancy over NDRs and decreased occupancy flanking NDRs. The latter change results in increased transcription of ncRNAs surrounding NDRs throughout the genome. Transcription factors and RNAPII are represented by green and blue objects.