Nature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1. Validation of CDK9-inhibitor treatment.

Transcription

1 Supplementary Figure 1 Validation of CDK9-inhibitor treatment. (a) Schematic of GAPDH with the middle of the amplicons indicated in base pairs. The transcription start site (TSS) and the terminal polyadenylation site (pa) are marked by arrows, exons by boxes and introns by lines. (b) qrt-pcr analysis of nascent RNA from GAPDH after treatment with KM05382 (KM) and DRB for the indicated time. The nascent RNA level is normalised to the 7SK RNA level. Error bars, s.e.m. (n = 3 biological replicates). (c) Ser2P and Ser5P ChIP levels related to pol II ChIP level (Fig. 1D) on GAPDH performed on HeLa cells untreated (Cont.) or treated with 100 M KM or DRB. Error bars, s.e.m. (n = 3 biological replicates).

2 Supplementary Figure 2 GRO-seq read processing and reproducibility. (a) Distribution of sequenced GRO-seq fragment sizes after pre-processing (elimination of fragments shorter than 20nt). (b) Number of read-pairs for each of the four GRO-seq samples remaining after subsequent processing steps. (c) Number of reads mapped to 5 ETS and 3 ETS of pol I-transcribed 45S pre-rrna (NR_046235), to the intron of the pol III-transcribed Leucine trna gene (Leu74) and to the gene-body of the pol II-transcribed GAPDH protein-coding gene (NM_002046), showing that only pol II transcription is affected by treatment. (d) Ratio of read-counts in these gene segments relative to the control (Cont.) after normalisation to 45S-5 ETS. (e,f) Metagene profile relative to the annotated transcription start site (TSS) (e) and terminal poly adenylation-cleavage sites (pa) (f) in two untreated (Cont.) HeLa samples. Only protein-coding transcripts longer than 5kb are included in this analysis. (g) The high level of correlation between the two repeats in untreated HeLa cells indicates good reproducibility of the GRO-seq procedure. Read counts are integrated in 10kb windows across the genome (Spearman correlation 0.90). (h) Metagene profile (as if each transcript was 10kb in length) of polymerase distribution in untreated HeLa cells with reads from both repeats merged. Notable features: a marked TSS proximal peak both in sense and antisense orientation; a marked sense-only peak downstream of the terminal pa site; depletion of antisense reads within the gene body.

3 Supplementary Figure 3 TSS-proximal checkpoints. (a) GRO-seq profile of DHX9 with treatment indicated on the UCSC genome browser. Forward strand reads are noted in blue and reverse strand reads in red here and in subsequent figures. The direction of sense transcription is marked by an arrow. (b) qrt-pcr analysis of pol II ChIP performed on HeLa cells untreated (Cont.) or treated with 100 M KM or DRB. Error bars, s.e.m. (n = 3 biological replicates). (c) Distribution of the positions of peaks of antisense signal (in 100bp windows) relative to the annotated TSS for each transcript. While for the majority of genes the peak of antisense transcription occurs upstream of the TSS, there is a subset of genes (larger after P-TEFb inhibition) with significant antisense transcription downstream of the TSS.

4 Supplementary Figure 4 KM and DRB inhibit transcription in the gene bodies without increasing transcription close to the TSSs of EIF2S3 and PLK2. (a) GRO-seq profiles of PLK2 in untreated (Cont.) and treated (KM, DRB) cells. (b) Top, gene schematic of PLK2. Bottom, qrt-pcr analysis of pol II, Ser5P and Ser2P ChIP performed on HeLa cells untreated (Cont.) or treated with 100 M KM or DRB and Ser2P and Ser5P relative to the pol II ChIP level. Error bars, s.e.m. (n = 3 biological replicates). (c) Top, gene schematic of EIF2S3. Bottom, qrt- PCR analysis of Ser5P ChIP, Ser2P and Ser5P relative to pol II ChIP level in untreated (Cont.) and treated (KM, DRB) cells. Error bars, s.e.m. (n = 3 biological replicates).

5 Supplementary Figure 5 CDK9 inhibitors cause premature termination of transcription close to pa sites. (a) The metagene profile of polymerase distribution in treated (KM, DRB) and untreated (Cont.) cells for transcripts longer than 50kb, shown as if each transcript would be 10kb in length. It demonstrates the signal recovery on long genes and loss of polymerase density downstream of the pa after treatment. (b) GRO-seq coverage of DHX9 with treatment indicated. (c) Top, gene schematic of DHX9. Bottom, qrt-pcr analysis of pol II ChIP and nascent RNA in untreated (Cont.) and treated (KM, DRB) cells. Error bars, s.e.m. (n = 3 biological replicates). (d) Top, gene schematic of KPNB1. Bottom, qrt-pcr analysis of pol II performed on HeLa cells untreated (Cont.) or treated with 1 M flavopiridol. Error bars, s.e.m. (n = 3 biological replicates). (e) Wild type (WT) and analog-sensitive (AS) CDK9 nucleotide and protein sequences. (f) Western blot analysis of extracts from HEK 293 cells transfected with Myc epitope-tagged CDK9AS and anti-cdk9 shrna (CDK9sh) and treated or not with 1-NA-PP1 as indicated. Antibodies used are noted on the right. Actin

6 serves as a loading control. (g) Left, Gene schematic of KPNB1. Right, qrt-pcr analysis of pol II ChIP performed on HEK 293 cells untreated (Cont.) or treated with 100 M KM or 10 M 1-NA-PP1. Error bars, s.e.m. (n = 3 biological replicates).

7 Supplementary Figure 6 Effect of KM and DRB on modification of the pol II CTD. (a) Ratio of qpcr analysis of Ser2P, Ser5P, Ser7P, Thr4P and Tyr1P ChIP to pol II ChIP in untreated (Cont.) and treated (KM, DRB) cells. For the probes pa-0.4, pa+1.4 and pa+2.6 after KM and DRB treatment, the level of pol II is too low to be used and values are only shown for the control sample. (b) qrt-pcr analysis of Ser2P, Ser5P, Ser7P, Tyr1P and Thr4P ChIP in untreated cells. The highest value of each ChIP has been normalised to 1 to facilitate comparison of CTD phosphorylation along KPNB1. (c) Western blot analysis of cell extract after drug treatment, using the indicated antibodies. -tubulin serves as a loading control.

8 Supplementary Figure 7 Effect of KM and DRB on recruitment of Pcf11 and Spt5 to KPNB1. Ratio of qpcr analysis of Pcf11 and Spt5 ChIP to pol II ChIP in untreated (Cont.) and treated (KM, DRB) cells. For probes pa-0.4, pa+1.4 and pa+2.6 after KM and DRB treatment, the levels of factors and pol II are generally very low and ratios are only shown for the control samples and Spt5 related to pol II on probe pa-0.4 after DRB treatment.

9 Supplementary Table Name Sequence of forward primer Sequence of reverse primer Amplified region GAPDH +119 AGGTGAAGACGGGCGGAGAGA GCGAACACATCCGGCCT +78/+160 (TSS) GAPDH +925 CTCGATGGGTGGAGTCGC CTAGGAAAAGCATCACCCGGA +881/+968 (TSS) GAPDH GGTGGTGAAGCAGGCGTCGGAGGGC GAGCCAGTCTCTGGCCCCAGCCACA +3348/+3534 (TSS) 7SK CTGATCTGGCTGGCTAGGCGGG GAAGACCGGTCCTCCTCTATCGG +11/+181 (TSS) 5 ETS rrna TGAGTGAGACGAGACGAGAC GAAGTCAACCCACACACGA +961/+1168 (TSS) Leu74 trna AGC TTG GCT TCC TCG TGT T TGTCAGAAGTGGGATTCGAAC +37/+105 (TSS) TRPM7-927 GATGGAGAGGGACGAGATGAAGG GACATACCTGTGCACCCATG -979/-876 (TSS) TRPM CTAGCGCCGGAGCTGAGTTAG TTCCCGATAGATGGCTACAGG +120/+225 (TSS) TRPM CGGTAAAGGAGAGACTTGGCA GGGACGGGTGTGCAGTTCGTTCT +334/+455 (TSS) TRPM GGGACGGGTGTGCAGTTCGTTCT CAGTTGCTGGCAGATAAGGGAA +614/+692 (TSS) TRPM GGAACCAAAAGGGAGGGATT CTGTCAGTCAGGATTTAAGTCGAAC +830/+966 (TSS) DHX GATAACCTCGGCCAACGCGAAG CATGTGACTGGAGTGTGGCGA +600/+720 (TSS) DHX GGTGGAAGGTAGTAGATCTGGA GGCTCACAGCAACGTCGGTC +878/+1020 (TSS) DHX GAAATGTAGGGGTAGGGTGTTGG GGGTGAGTTAGTGCCACAGTTGGATG +1388/+1611 (TSS) DHX CATTCACCATGAGGGCTTGTTGTTTAAT TTCCCTTCGCCAAATGGATGACCA +2066/+2184 (TSS) PLK AAGTGTCTCCTCTGTACCAGGA GGAATCATGACCAGGAAATGTACGG -1440/-1332 (TSS) PLK ACCGGGGTGTTGGGTGCTAGT ATAGTCCGCAAAAGCTCCATG +185/+321 (TSS) PLK AGTGCTGGGAAAGGTGACAAGCGG ATCGGACCCCCGAAAAACCCGGAA +559/+689 (TSS) PLK GTTCCAACTCCCTCCATAAAGCTC TTGAGGAATGGAGGAGGGTGGACA +916/+1061 (TSS) EIF2S AACCAGCGAACTTCAGACGCT GTCCCCAGCTTGTTCCCAGAGA -480/-360 (TSS) EIF2S GAGAAGCTGGAGTGACTCTAGG CACTGACTAGTCCCAATACC +264/+406 (TSS) EIF2S GGTGTCCTGTCTGCATTACTT CACAACAGACTACACCTCCAC +610/+714 (TSS) EIF2S GATGGTGGCAAGATGTAGATAGCA CGTCAACTTGGTAACATCCTGCAATG +808/+917 (TSS) EIF2S TACAGGCCTTGAACTACTGC CTTAGCATAGGTTGTTCGGAGG +1591/+1679 (TSS) KPNB1 TSS+0.9 AGGGAAGGGAAGTTAGGTTGCG GAGCTTCCACATGGCCCCTA +900/+986 (TSS) KPNB1 TSS+5.3 GCAAAGCAGGATACTAAGTGATCCGA GAAGCCACAATTGACCTAGAGC +5234/+5405 (TSS) KPNB1 TSS+20.2 TGCAAGAGCCAGTGGGAACACTT CCTCTACTCAGCAATGATACTTC / (TSS) KPNB1 pa-4.8 CTGAGGAAACTGAAGAACCAAG GAAGGCAGTGCTTGCCAGAAT -4862/-4746 (end) KPNB1 pa-2.9 GAGGAGTGTGCACGGATGCTGAA CCAAGATGGCCGATGTTATGG -3030/-2855 (end) KPNB1 pa-0.4 TAGTTACCGTCTGCTTGGGAAGATG CCTCTGACAGCAAGTCCAACATT -510/-330 (end) KPNB1 pa+1.4 GACTCATCACACCAAGGTCAC GATAGTGCTGGGAAGGAAATGG +1320/+1490 (end) KPNB1 pa+2.6 GTACATCTCAGCTTTGGCATATG GCCCAGAACATAGCAGGCATTGC +2587/+2728 (end) KPNB1 pa+4.1 GTTTCACCGTGTTAGCCAGGATGG CCACAGCCATGTTCATTTCTGC +4053/+4217 (end) KPNB1 pa CAGTCTGTGATGGCATTTAAG TCTGACTTTCAAACCATTTCACCTCC -114/+77 (end) DHX CCTGCCTCAAGATGATCTCC GCTAGCTACAGCTGTACAGC +1028/+1208 (end) DHX GAAAGTGCTTCCACATAGCCATGG CTGGATTCCCACTTACAGCTATGAC +1568/+1740 (end) DHX9-122 GGC ATG CTA TGT GTT ACG TG CGAGGTCAACAAACAAACTACACGGC -272/+29 (end) Supplementary Table. Sequences of primers used and their position relative to the TSS or the end of gene (end).