1. Volume 21, Issue 9, Pages 2433-2446 (November 2017)
Control of Gene Expression in Senescence through Transcriptional Read-Through of Convergent Protein-Coding Genes 
Lisa Muniz, Maharshi Krishna Deb, Marion Aguirrebengoa, Sandra Lazorthes, Didier Trouche, Estelle Nicolas 
Cell Reports 
Volume 21, Issue 9, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2433-2446DOI: (10.1016/j.celrep.2017.11.006)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Occurrence of Antisense RNAs Generated by Regulated Transcriptional Read-Through in Senescence (START RNAs)
(A) Schematic representations of antisense RNA generated by transcriptional read-through (START RNA) from a convergent gene (the forward gene) located on the plus strand (+) of DNA (left) or on the minus strand (−) of DNA (right). The START RNA is antisense to the other gene of the convergent gene pair (reverse gene).
(B) Proliferative and senescent WI38 hTERT RAF1-ER cells were subjected to two independent strand-specific RNA-seq experiments (RNA-seq #1 and RNA-seq #2). Metadata analyses of the 2 RNA-seq datasets in proliferation (PROLIF, green) or in senescence (SEN, red) at the 91 START loci are shown. Each gene and intergenic regions were divided into 20 equal regions. For each of these regions, the mean of the normalized number of aligned reads per base was computed for each gene, and the median within the gene population is plotted. The forward strand and the reverse strand of RNA-seq datasets are shown with a full lane and a dashed lane, respectively.
(C) Boxplots showing the log2 of the expression change in senescence, log2(sen/prolif), of the forward genes and of different analyzed regions in the 91 START RNAs: the antisense (AS) parts, the entire read-through (RT) transcript domains, corresponding to the entire START RNAs, as well as their first (read-through, part 1) and second (read-through, part 2) halves. Two independent datasets are shown. The read-through transcript domains and the AS parts of the 91 START RNAs are significantly more increased in senescence than their forward genes (paired Wilcoxon test). Note that the second halves of START RNAs are significantly more increased in senescence than the first halves (paired Wilcoxon test).
See also Figures S1 and S2 and Tables S1 and S2.
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4. Figure 2
Analysis of START Expression at the ARHGAP18/LAMA2 and KIAA1919/REV3L Loci
(A) Total RNA was extracted from cells in proliferation (PROLIF) or in senescence (SEN) and subjected to strand-specific RNA-seq experiments. For the (+) and (−) DNA strands at the ARHGAP18/LAMA2 and KIAA1919/REV3L loci, strand-specific RNA-seq tracks (RNA-seq#1) show the density of aligned reads normalized by the total number of aligned reads multiplied by 100 millions. RefSeq genes (hg38) are also shown. The red dotted arrows show the boundaries of the START RNAs found by the algorithm.
(B) The total number of reads from strand-specific RNA-seq data (RNA-seq#1) in the indicated genomic regions in proliferative and senescent cells were calculated for the two loci. The log2 of the SEN/PROLIF value is plotted. The chromosome strand of the analyzed region is annotated. The values for the forward gene (ARHGAP18 or KIAA1919), the intergenic region of the read-through (RT) transcript, the read-through entire domain, the antisense (AS) part of the read-through (AS part to LAMA2 or REV3L), and the reverse gene (LAMA2 or REV3L) are shown.
(C) WI38 hTERT RAF1-ER cells were induced (+ 4-HT) or not induced (- 4-HT) to enter senescence for 3 days. Total RNA was extracted and subjected to random qRT-PCR using the indicated primers: e, exon; e-e, exon-exon junction; and down, downstream of the TTS of the gene. Data were normalized to GAPDH mRNA expression and calculated relative to 1 in proliferative cells for each experiment. Data are indicated as means ± SD from 3 independent experiments.
(D) Same as in (C), except that total RNA was subjected to strand-specific qRT-PCR and analyzed using the indicated primers (i, intron).
See also Figure S3.
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5. Figure 3
ARHGAP18 and KIAA1919 START RNAs Are Generated by Transcriptional Read-Through
(A) Schematic representation of the ARHGAP18/LAMA2 and KIAA1919/REV3L loci with the location of siRNAs (orange) and PCR primers (purple) used in Figures 3 and 4.
(B) Senescent cells were transfected using an siRNA targeting ARHGAP18 exon 1 (ARHG e1) or control (Ctrl). 72 hr after transfection, total RNA was extracted and subjected to random qRT-PCR using the indicated primers (left) or analyzed by strand-specific qRT-PCR to monitor the expression of the region antisense to LAMA2 (right). Data were normalized to GAPDH mRNA expression and calculated relative to 1 in siRNA Ctrl-treated cells for each experiment. Data are indicated as means ± SD from 3 independent experiments.
(C) Same as in (B), except that cells were transfected using an siRNA targeting the read-through region (ARHG R-th) in the intergenic region between ARHGAP18 and LAMA2 (38.7 kb downstream of the TTS of ARHGAP18).
(D) Same as in (B), except that cells were transfected using an siRNA targeting KIAA1919 read-through region (KIAA R-th, 2.5 kb downstream of the TTS of KIAA1919), and strand-specific qRT-PCR was performed to monitor the expression of the region antisense to REV3L (right panel).
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6. Figure 4 START RNAs Repress the Genes to which They Are Antisense
(A) Total RNAs from senescent cells transfected with an siRNA targeting ARHGAP18 exon 1 (ARHG e1), targeting the read-through region (ARHG R-th) or control (Ctrl) were extracted and analyzed by strand-specific qRT-PCR for LAMA2 (pre-mRNA/mRNA) sense expression (e64). LAMA2 mRNA expression (e64-65) was analyzed by random qRT-PCR using primers in an exon-exon junction. Data were normalized to GAPDH mRNA expression and calculated relative to 1 in siRNA Ctrl-treated cells for each experiment. Data are indicated as means ± SD from 3 independent experiments.
(B) Same as in (A), except that cells were transfected using an siRNA targeting KIAA1919 read-through region (KIAA R-th). REV3L (pre-mRNA) sense expression was monitored by strand-specific qRT-PCR in an intron (left panel, i32) or by random qRT-PCR using primers in an intron located far away from the read-through antisense transcript (right panel, i2). The spliced REV3L mRNA was assessed by random qRT-PCR using primers in an exon-exon junction (right panel, e32-e33).
(C) Total RNAs from proliferative or senescent cells were extracted and subjected to strand-specific RNA-seq experiments. For the 91 loci harboring START RNAs, the log2 of the variation in senescence (log2(sen/prolif)) of the expression of the reverse genes was computed and represented as a boxplot for both RNA-seq experiments (#1 and #2). Note the significant decrease (Wilcoxon test) in the expression of the reverse gene population compared to all expressed genes to which an antisense transcript is increased in senescence (2,135 and 1,349 genes in the first and second RNA-seq replicates, respectively).
(D) Senescent cells were transfected using the indicated siRNAs. Transfected cells were then subjected to a colony formation assay in duplicate. Colony number was counted twice in each plate and calculated relative to 1 for the control siRNA. Data are indicated as means ± SD from four entirely independent experiments.
See also Figure S4.
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7. Figure 5
The Increase in ARHGAP18 START RNA Levels in Senescence Is Transcriptional
(A) Total RNA from cells in senescence (SEN) and proliferation (PROLIF) were analyzed by qRT-PCR using the indicated primers at different time points following actinomycin D treatment. The levels of RNA were normalized to those of GAPDH mRNA and then normalized to 100% at time 0, for each experiment. Data are indicated as means ± SD from 3 independent experiments (logarithmic scale).
(B) Senescent (+4-HT) and proliferative (−4-HT) cells were subjected to an EU pulse labeling of 1 hr, after which nascent RNAs were captured. Nascent RNAs were then analyzed by qRT-PCR using the indicated primers. Data were normalized to GAPDH mRNA expression and calculated relative to 1 in proliferative cells. A representative experiment out of two is shown (means ±SD from the qPCR sample triplicates).
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8. Figure 6
Pol II Elongation Rate Increases in Senescence after the Poly(A) Site of the ARHGAP18 and KIAA1919 Genes
Total RNA from cells in senescence (SEN) and in proliferation (PROLIF) were analyzed by qRT-PCR at different time points following flavopiridol treatment. The values for ARHGAP18 mRNA (e1) and GAPDH pre-mRNA (i1) (as controls) or ARHGAP18 intron 3-exon 4 junction (i3-e4), ARHGAP18 intron 14 (i14), ARHGAP18 read-through (1 kb and 38.3 kb downstream of the TTS of ARHGAP18 gene), and KIAA1919 read-through (measured 22.3 kb downstream of the TTS of KIAA1919) are shown. The levels of RNA are normalized to those of GAPDH mRNA and then normalized to 100% at time 0 for each experiment. Data are indicated as means ± SD from 3 independent experiments (logarithmic scale). When the SD is higher than the mean (for ARHGAP18 i3-e4 in proliferation, 2 hr), only the upper error bar is shown.
See also Figure S5.
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9. Figure 7
ARHGAP18 and KIAA1919 Read-Through RNAs Are Repressed by H2A.Z in Proliferative Cells
(A) WI38 hTERT RAF1-ER cells were induced, or not induced, to enter senescence by the addition of 4-HT for 3 days. Total cell extracts were analyzed by western blot using the indicated antibodies. One representative experiment out of 7 is shown.
(B) Proliferative cells were transfected using the indicated siRNAs. 72 hr following transfection, total RNA was prepared and subjected to random qRT-PCR using the indicated primers. Data were normalized to GAPDH mRNA expression and calculated relative to 1 in siRNA control (Ctrl)-treated cells for each experiment. Data are indicated as means ± SD from 3 independent experiments.
(C) Same as in (B), except that the H2A.Z#2 siRNA was used, and one representative experiment out of two is shown (means ± SD from the qPCR sample triplicates).
(D) Same as in (B), except that total RNAs were prepared, depleted of rRNA, and sequenced. For the 91 START loci, the log2 of the variation upon H2A.Z depletion (log2(siH2A.Z/siCtrl)) of the expression of the forward genes and of the read-through domains (START RNAs) were computed. Note the significant increase (paired-Wilcoxon test) in the expression of the START RNAs compared to the forward genes upon H2A.Z depletion. The RNA-seq replicate #2 is shown.
(E) Cells in proliferation (PROLIF) or senescence (SEN) were subjected to ChIP experiments using H2A.Z antibodies, using H3 antibodies, and without antibody (NA). The indicated sequences were quantified by qPCR in the inputs and the immunoprecipitates. The enrichment of the indicated sequence in the H2A.Z immunoprecipitate was calculated relative to the input and normalized to the enrichment in H3 immunoprecipitate. The ratio between the indicated sequence and a control sequence (GAPDH e1) was then performed and calculated relative to 1 in the H2A.Z ChIP in proliferative cells for each experiment. Data are indicated as means ± SD from 4 or 5 independent experiments.
(F) Proliferative and senescent cells were subjected to an H2A.Z ChIP-seq analysis (ChIP-seq replicate #2). Boxplots show the log2 of the variation in senescence (log2(sen/prolif)) of H2A.Z occupancy at the forward gene TSS and the intergenic regions for the loci harboring START RNAs with an intergenic region >1 kb (to limit the analysis to intergenic regions because of the ChIP resolution of around 500 bp [69/91]). Note that H2A.Z occupancy significantly decreased in senescence among this population on these two regions. The p values of the difference to 0 are 5.33 ⋅ 10−13 for TSS (Student’s t test) and 1.41 ⋅ 10−12 for intergenic regions (Wilcoxon-Mann-Whitney test).
See also Figures S6 and S7.
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