B
TERRA-CHIRT d0 ES0 1 2 3 4 5 6 Log2 coverage TERRA-CHIRT Rep1
Person’s r=0.69
Name of library Total reads
Reads after
ES_d0 TER-AS 28932181 23905938 22631527 94.67
ES_d0 TER-sense 46282449 44747817 43894751 98.09
ES_d0 TER (no RNAseH) 41852461 34807415 32453318 93.24
ES_d0 input 45396054 41983022 41545697 98.96
Replicate 1
Name of library Total reads
Reads after
ES_d0 TER 14094905 12515985 10725993 85.70
ES_d0 TER RNase A 13627224 12411472 10985881 88.51
ES_d0 sense 14950599 13133452 10864427 82.72
ES_d0 sense RNase A 13903763 12050175 10459053 86.80
ES_d0 TER no Rnase H 13666488 10053384 8013263 79.71
Replicate 2 CHIRT
CHIRT Number of TERRA
foci in nucleus
Number of TRF2 foci colocalized with TERRA 0
20 40 60
% of TERRA colocalized with TRF2
ES cells
Analysis of genomic regions with telomeric repeats
5'UTR 3'UTR Coding exons
TERRA-CHIRT normalized to input 2
TERRA-CHIRT normalized to sense0
TERRA-CHIRT
TERRA-CHIRT normalized to no-RNaseH 0 r=0.93
(legend on next page)
Figure S1. TERRA CHIRT-Seq Statistics, Related toFigure 1
(A) TERRA colocalization counts with TRF2 for the data inFigure 1C. 414 out of 2014 TERRA foci (19.7%) were colocalized with TRF2 in mouse ESCs. n = number of counted nuclei.
(B) RNA slotblot analysis showing that TERRA-AS specifically captured TERRA RNA by CHIRT. Total RNA was extracted from beads after probe hybridization without RNase H elution.
(C) Scatterplot comparing Log2 coverages of biological replicates for TERRA CHIRT-seq analysis in ESCs. Pearson’s r was shown. Replicate 1 (Rep1) was normalized with input. Replicate 2 (Rep2) was normalized to RNaseA pre-treated control.
(D) Read statistics for two biological replicates of the TERRA CHIRT-seq analysis.
(E) Scatterplot analysis comparing log2 coverages of TERRA CHIRT normalized using different methods. Pearson’s r shown.
(F) CEAS analysis shows significant over-representation of introns and noncoding regulatory regions. Exons are under-represented. ***p < 0.001; *p < 0.05 (one-sided binomial test). The genome reference was obtained from the CHIRT input.
(G) CEAS analysis: Left panel, Pie chart showing relative representation of various genomic regions when the CHIRT analysis excludes genomic regions con-taining telomeric repeats. Right panel, Pie chart showing relative representation of various genomic regions when the CHIRT analysis is performed on genomic regions containing telomeric repeats.
(H) CHIRT-seq tracks showing TERRA enrichment at the ends of various chromosomes in female ESCs. Different normalization methods reveal similar results (TERRA/input; TERRA/no RNase H; TERRA/sense). Note: TERRA-Sense CHIRT did not resemble TERRA-AS CHIRT.
-15-10 -5 0 5 10 15
Log2 ΔFPKM of TERRA KD Rep1
B
Flnb Dnase 1l3 Abhd6 Chr14: 7,820-8,060 kb Chr4: 85,200-85,450 kb
[0-300] of TERRA KD Rep2
Log2 ΔFPKM
Upregulated genes Downregulated genes
Sense
(legend on next page)
Figure S2. RNA-Seq Analysis after TERRA Knockdown by LNA Gapmers, Related toFigure 2 (A) RNA FISH confirming TERRA depletion after 1, 6, and 24 hr of LNA treatment in ESCs.
(B) Scatterplots comparing log2 FPKM values show good correlation between biological replicates of RNA-seq in both Scr KD and TERRA KD in ESCs. Pearson’s r is shown.
(C) CHIRT-seq tracks (red) for TERRA binding sites near differentially upregulated genes after TERRA KD in ESCs. RNA-seq coverage is FPM (fragments per million)-normalized and tracks are shown in blue.
(D) CHIRT-seq tracks (red) for TERRA binding sites near differentially downregulated genes after TERRA KD in ESCs. RNA-seq coverage is FPM (fragments per million)-normalized and tracks are shown in blue.
(E) Quantitative RT-qPCR of Nfib and Erdr1 expression after Scr, sense, or TERRA LNA knockdown.
(F) RNA-seq analysis of sense LNA knockdown controls show largely non-overlapping changes in comparison to TERRA-specific knockdown.
Count P-Value Benjamini
TOR signaling 3 1.1E-3 3.6E-1
cellular response to corticotropin-releasing hormone stimulus 2 1.1E-2 8.9E-1
protein localization 3 1.1E-2 7.8E-1
positive regulation of transcription from RNA polymerase II promoter 7 1.6E-2 8.1E-1 negative regulation of transcription from RNA polymerase II promoter 6 1.6E-2 7.4E-1
negative regulation of hormone secretion 2 2.1E-2 7.7E-1
negative regulation of pri-miRNA transcription from RNA polymerase II promoter 2 2.1E-2 7.7E-1
post-embryonic development 3 2.4E-2 7.6E-1
negative regulation of cAMP-dependent protein kinase activity 2 3.1E-2 8.1E-1
neuron maturation 2 3.1E-2 8.1E-1
clathrin coat assembly 2 3.1E-2 8.1E-1
GO Term
Downregulated genes
A
Count P-Value Benjamini
heart development 10 5.8E-5 4.5E-2
protein phosphorylation 12 1.5E-3 4.5E-1
positive regulation of telomere capping 3 4.9E-3 7.3E-1
organ morphogenesis 5 5.8E-3 6.9E-1
negative regulation of MAPK cascade 3 6.8E-3 6.7E-1
regulation of JNK cascade 3 6.8E-3 6.7E-1
protein glycosylation 5 7.3E-3 6.2E-1
protein ADP-ribosylation 3 7.6E-3 5.8E-1
transcription elongation from RNA polymerase II promoter 3 9.1E-3 6.0E-1
response to calcium ion 4 1.0E-2 6.0E-1
Upregulated genes
B
GO Term
GENE NAME
epidermal growth factor receptor(Egfr) insulin-like growth factor I receptor(Igf1r)
tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase(Tnks)
Upregulated genes_Telomerase pathway
C
Figure S3. GO Analysis of Upregulated and Downregulated Genes following TERRA Depletion, Related toFigure 2 Enriched pathways are identified by DAVID bioinformatics resources. p values determined by Fisher Exact test. Benjamini FDR shown.
A
Log2 fold enrichment (TERRA/Luc)Log2 fold enrichment (TERRA/U1)
B
0 1 2 3 4 5 6
TERF1 4732456N10Rik ACD POT1 PAXIP1 RTEl1 COIL Rbm45 TINF2 CTC1 CENPC STN1 SETD2 POT1B RALY INCENP HBB-B2 MIS18BP1 HNRNPC ATRX Borealin CASC5 CELF1 ZNF106 SMC6 ORC1 MYEF2 TARDBP
0 1 2 3 4 5 6
TERF1 4732456N10Rik ACD POT1 RTEL1 TINF2 PAXIP1 POT1B CTC1 STN1 CENPC RBM45 INCENP MIS18BP1 ATRX HBB-B2 HBA AURKB Borealin COIL PML AGR3 RALY HNRNPC S100A4 CASC5 SETD2 RPA1 SRP68 ORC1
C
-1 0 1 2 3 4 5 6
TERRA_avg/
Luciferase_avg Sense_avg/
Luciferase_avg U1_avg/
Luciferase_avg 0
1 2 3 4 5
PAXIP1 RTEL1 ATRX PML SMC6 SMC5 FANCA SMARCAL1 BLM COIL CTC1 STN1
Log2 fold enrichment Log2 fold enrichment
TRF1 POT1BTINF1POT1TPP1
Shelterin complex Alternative lengthening of telomeres (ALT) and other telomeric functions
Figure S4. TERRA iDRiP Proteomics, Related toFigure 3
(A) Top 30 candidates of TERRA iDRiP proteomics with the highest enrichment over the luciferase RNA capture are shown.
(B) Top 30 candidates of TERRA iDRiP proteomics with the highest enrichment over the U1 RNA capture are shown.
(C) Shelterin complex and telomere function associated proteins are enriched in TERRA iDRiP proteomics but not in the sense-probe or the U1 RNA proteomics.
The intensities of indicated proteins were normalized to luciferase RNA iDRiP proteomics. Log2 fold enrichment was shown as the y axis.
B
ChIP-seq TERRA
CHIRT-seq ATRXSMC1H3K9 me3PolII S5 RNA-seq Scr KDTER KD
Fyco1 Xcr1
[0-50]
[0-40]
[0-50]
[0-2]
[0-1000]
[0-1]
[0-1]
Lphn2 [0-0.5]
[0-0.5]
[0-50]
[0-40]
[0-50]
[0-2]
[0-1000]
A
ChIP-seq TERRA
CHIRT-seq ATRXSMC1H3K9 me3PolII S5 RNA-seq Scr KDTER KD
Scr KD TERRA KD
(TTAGGG)n GA-rich TERRA & ATRX common site
−3000−2000−1000 01000 2000 3000
0.070.11
−3000−2000−10000 1000 2000 3000
0.120.22
−3000−2000−10000 1000 2000 3000
0.270.31
−3000−2000−10000 1000 2000 3000
0.050.30
−3000−2000−1000 0 1000 2000 3000
0.140.24
−3000−2000−1000 01000 2000 3000
0.230.29
−3000−2000−1000 01000 2000 3000
0.030.040.08
−3000−2000−10000 1000 2000 3000
0.130.18
−3000−2000−10000 1000 2000 3000
0.240.34 H3K9me3 H3K27me3
H3K4me3 H3K36me3
POL-lI S5EZH2 SMC1 CTCF
ATRX
Distance from TERRA peak (bp)
Relative coverage
C
Figure S5. Gene Expression of ATRX-TERRA Target Genes after TERRA KD, Related toFigure 4
(A) Metasite analysis as described inFigure 4A but with telomere-bound TERRA peaks subtracted from analysis. ChIP-seq coverage of indicated epitopes (y axis) relative to averaged TERRA-binding site (x = 0).
(B) TERRA depletion results in Lphn2 and Fyco1 downregulation. Genome browser shot of two examples of TERRA targets, with RNA-seq, CHIRT-seq, and ChIP-seq data displayed.
(C) Heatmap shows the downregulation genes (green, log2 < 0.5 of delta FPKM) and the upregulation (red, log2 > 0.5 of delta FPKM) genes for ATRX/TERRA target after TERRA depletion.
0 1000 2000 3000
-1000 4000
0 0.2
0.1
0.2 0.8
0 1000 2000 3000
-1000 4000
0 1000 2000 3000
-1000 4000
0.05 0.25
Downregulated Upregulated
Relative distance (bp)
TSS TTS
ATRX ChIP-seq
H3K4me3 ChIP-seq
CTCF ChIP-seq Relative ATRX bindingRelative H3K4me3 coverageRelative CTCF binding
Figure S6. ATRX Is Enriched near the TSS of Genes Sensitive to TERRA Depletion, Related toFigure 4K
Relationship of ATRX, H3K4me3, and CTCF coverage profiles shown in metagene plots for genes that are upregulated (red) or downregulated (blue) after TERRA depletion. Note that ATRX coverage is enriched near the TSS of genes that are downregulated when TERRA is depleted. This trend was not observed for other epitopes, including H3K4me3 and CTCF.
G A RepA III-IV
G A U1 30nt TERRA 30nt
G ATRX
dsDNA ssDNA
[60nM]
[0.6- 60nM]
RNA [0.2nM]
B
U Protein
[60nM]
[0.6- 60nM]
RNA EMSA TERRA KD Scr KD
Telomeric DNAATRX
3 µm
* *
* * *
3 µm ATRX Telomeric DNADAPIDAPI Telomeric DNA
A
B
Figure S7. TERRA-ATRX Interactions Are Not Competed Away by DNA, Related toFigure 5
(A) Competition assay: TERRA RNA shifted by ATRX cannot be competed away by increasing concentrations (0.6, 6, 60nM) of dsDNA (30bp) or ssDNA (30nt) of corresponding TERRA sequence (30nt). RNA EMSA with 0.2 nM RNA probes from indicated transcripts (top) and 60nM GST (G, control) or ATRX (A) protein.
Bound probe (shifted), B. Unbound, U.
(B) Additional examples of TERRA depletion (6hr knockdown) causing ATRX dispersal from pericentric heterochromatin and relocalization to telomeres in ESCs.
Immuno-DNA-FISH staining for ATRX and telomeric DNA reveals large ATRX foci (red) on pericentric heterochromatin (dense DAPI staining) in control cells.
TERRA depletion cells show dispersed ATRX signals that coincide with telomeric DNA (green). Asterisks are ATRX foci at pericentric heterochromatin. Arrow-heads are ATRX foci at telomeres.