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The non-coding snRNA 7SK controls transcriptional termination, poising, and bidirectionality in embryonic stem cells

Gonçalo Castelo-Branco15, Paulo P Amaral1, Pär G Engström26, Samuel C Robson1, Sueli C Marques5, Paul Bertone234 and Tony Kouzarides1*

Author Affiliations

1 The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

2 European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SD, UK

3 Genome Biology and Developmental Biology Units, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany

4 Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

5 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet,SE-17177 Stockholm, Sweden

6 Present address: Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Box 1031, SE-17121 Solna, Sweden

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Genome Biology 2013, 14:R98  doi:10.1186/gb-2013-14-9-r98

Published: 17 September 2013

Additional files

Additional file 1: Figure S1:

(a) Quantitative reverse transcription (qRT)-PCR analysis of 7SK total RNA levels in two independent experiments in which embryonic stem cell (ESCs) were nucleofected with antisense oligonucleotides (ASOs) targeting 7SK at a position near the 5′ or 3′ end of the RNA (7SK 5′ or 7SK 53′ ASO). Error bars represent standard error of the mean (SEM) for qPCR technical replicates. (b) qRT-PCR analysis of Dll1 total RNA levels when ESCs were nucleofected with 7SK 5′ and 3′ ASOs. ESCs were replated after nucleofection and collected after 6 hours. Error bars represent SEM for qPCR technical replicates. (c) qRT-PCR analysis of 7SK, Dll1, Olig2, and Hexim1 total RNAs in ESCs after switch to 2iLIF media for several passages. (d) qRT-PCR analysis of Pou5f1 mRNA in ESCs 6 hours after nucleofection with 7SK 3′ ASO. ESCs were grown in serum (Ser-Ser) or 2iLIF media (2i-2i), or switched from 2iLIF to serum media after nucleofection (2i-Ser). Error bars represent SEM from two independent experiments. (e) qRT-PCR analysis of Pou5f1 nascent RNA in ESCs 6 hours after nucleofection with 7SK 3′ ASO. Error bars represent SEM from three independent experiments. (f) Sample preparation workflow for directional RNA sequencing (RNA-seq). Mouse ESCs were transfected with ASOs, and total RNA was extracted after 6 hours. Two independent experimental sets were used. Total RNA samples were treated with DNAse and depleted for ribosomal RNAs, but not enriched for polyadenylated RNAs. After RNA fragmentation and 5′ and 3′ end polishing, adapters were ligated to the RNAs, in accordance with the instructions of the TruSeq Small RNA sample prep kit (Illumina). The amplified DNA was clustered and run in an Hi-Seq instrument (Illumina) to obtain single-end reads of 50 nucleotides in length. Bioinformatic analysis was performed as described in the Materials and Methods section. (g) Breakdown of the number of sequenced reads per sample in the directional RNA-seq, including number of reads mapped to the mouse genome.

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Additional file 2: Figure S2:

(a) Ensembl genome browser screenshot showing normalized RNA-seq read coverage (mean of the two biological replicates) at the Nr4a2 (Nurr1) locus. The plus (green) and minus (blue) strand reads are displayed in separate tracks. (b) Gene Ontology terms associated with 7SK-regulated genes. Enrichment P-values were adjusted using the Benjamini and Hochberg multiple testing correction method.

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Additional file 3: Table S1:

Genes with altered expression after 7SK knockdown with two different antisense oligos.

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Additional file 4: Table S2:

All genes with altered expression after 7SK knockdown.

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Additional file 5: Figure S3:

Box plots and scatter plot depicting log2 fold changes measured by RNA sequencing (RNA-seq) after 7SK knockdown in mouse ESCs, by counting reads over exons and introns. Of the 438 genes found to be upregulated after 7SK knockdown, only those with introns are shown (397).

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Additional file 6: Figure S4:

Density scatter plots of normalized read counts for protein-coding genes and surrounding regions. Read counts from experiments in which embryonic stem cell (ESCs) were nucleofected with antisense oligonucleotides (ASOs) targeting the 5′ and 3′ parts of 7SK (y-axis) were plotted versus counts for ESCs nucleofected with scrambled control ASOs (x-axis), to illustrate the overall change in expression levels after 7SK depletion. Color intensity indicates the density of data points. Note the increased read coverage in upstream and downstream regions in 7SK-depleted samples. Read counts were normalized by the trimmed mean of M-values (TMM) algorithm (see Materials and Methods) and incremented by a pseudocount of 1 to enable visualization on a logarithmic scale. Upstream and downstream 5 kb regions were selected as described in Materials and Methods to avoid inclusion of segments from neighboring genes.

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Additional file 7: Table S3:

Coordinates of genes with failed transcriptional termination regions.

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Additional file 8: Figure S5:

(a) Gene-density analysis for failed termination genes. Gene density was computed as the number of unique genes (protein-coding genes and long intergenic non-coding RNAs (lincRNAs) greater than 1 kb long) within a window of +/−100 kb around the end position (final polyadenylation site) of each gene. The resulting distributions are shown for the 1,894 failed transcriptional termination genes (red) versus all other genes (black). In both sets, the majority of genes were found to have 0 to 10 genes within the 200 kb window (failed transcriptional termination genes: mean = 5.949, median = 5; other genes: mean = 5.391, median = 4). (b) Box plot depicting log2 fold changes by RNA sequencing (RNA-seq) after 7SK knockdown of downstream sense RNAs and their associated genes in mouse embryonic stem cells (ESCs). (c) Gene Ontology terms associated with 7SK-regulated genes, after background correction. Enrichment P-values were adjusted using the Benjamini and Hochberg multiple testing correction method. (d) Published poly(A)-negative whole-cell RNA-seq data from human ESCs (ENCODE) showed the presence of upstream divergent RNAs (udRNAs) (purple box). The plus (green) and minus (blue) strand reads are displayed in separate tracks.

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Additional file 9: Table S4:

Genes with altered expression after 7SK knockdown with antisense oligos and local background adjustment.

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Additional file 10: Table S5:

Upstream divergent RNA (udRNA) transcription units.

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Additional file 11: Figure S6:

(a) Venn diagram showing the overlap between upstream divergent RNAs (udRNAs) and antisense transcription start site (TSS)-associated (TSSa) RNAs at the TSS. (b) Venn diagram showing that 44.69% (274 of 613) udRNAs overlapping with divergent long non-coding RNAs (lncRNAs) were also upregulated after 7SK knockdown. (c) Venn diagram showing the overlap between genes with failed termination after 7SK knockdown (‘hotspot’ genes) and 7SK-regulated udRNAs. (d) Quantitative reverse transcription (qRT)-PCR analysis of Hexim1 total RNA, and Dll1 nascent RNA, 6 hours after nucleofection of embryonic stem cell (ESCs) with scrambled 7SK 3′ antisense oligonucleotides (ASOs) targeting the 3′ segments of 7SK, in the presence or absence of I-BET151. Error bars represent standard error of the mean (SEM) from two to three independent experiments. (e) Box plot depicting log2 fold changes measured by RNA sequencing (RNA-seq) after 7SK knockdown of udRNAs and their associated genes in mouse ESCs, using either 7SK 5′ or 7SK 3′ ASO data for udRNA detection.

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Additional file 12: Table S6:

Gene Ontology analysis of upstream divergent RNAs (udRNAs).

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Additional file 13: Figure S7:

(A) Quantitative reverse transcription (qRT)-PCR analysis of 7SK and Olig2 total RNA, and Sox9 mRNA levels after nucleofection of neural stem cells (NSCs) with 7SK 5′ and 3′ antisense oligonucleotides (ASOs), compared with scrambled and green fluorescent protein (GFP) ASOs (control; CTRL). NSCs were replated after nucleofection, and collected after 6 and 24 hours. Error bars represent standard error of the mean (SEM) for two independent experiments. (B, C) qRT-PCR analysis of (B)Hes1, Irx2, and Nr2a4 nascent RNA and (C)Hes1 and Rbm34 udRNA after nucleofection of NSCs with 7SK 3′ ASOs compared with scrambled ASO (CTRL). NSCs were replated after nucleofection, and collected after 6 hours. Error bars represent standard deviation (SD) of qPCR technical replicates. (D, E) qRT-PCR analysis of (D)7SK, Sox2, Hexim1, and Olig2 total RNA, and Dll1, CNP, and MBP nascent RNA and (E)Rbm34, hnRNPL, and Hes1 udRNA, Sox8OT (AK079380) total RNA, and Sox10OT (Gm10863) spliced RNA after lipofection of Oli-neu oligodendrocyte precursor cells (OPCs) with 7SK 3′ ASOs compared with scrambled ASOs (CTRL). OPCs were collected after 6 and 24 hours. Error bars represent SEM for three independent experiments.

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Additional file 14: Table S7:

Sequence of quantitative reverse transcription (qRT)-PCR primers and antisense oligonucleotides.

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