a 33,312 b rep 1 rep 1 # 44,325 rep 2 # 44,055 [0-84] rep 2 [0-84] 1810043G02Rik Pfkl Dnmt3l Icosl rep 1 [0-165] rep 2 [0-165] Rps14 Cd74 Mir5107 Tcof1 rep 1 [0-69] rep 2 [0-68] Id3 E2f2 Asap3 rep 1 [0-141] rep 2 [0-141] Ndel1 Rnf222 Rpl26 Odf4 Arhgef15 Slc25a35 Pfas Ctc1 Aurkb 2310047M10Rik Vamp2 Per1 Hes7 Aloxe3 Supplementary Figure 1 Consistency of ChIP-seq in mtecs. a) Venn diagram displaying number of overlapping peaks identified in two independent experiments. b) Exemplar genome browser views of two independent ChIP-seq experiments in B6. +/+ mtec hi.
Supplementary Figure 2 The mtec super-enhancer repertoire. a) Histogram depicting size density of super-enhancers (s) across mtec genome. Dotted yellow line depicts the median length for all super-enhancers. Dotted black line shows a smoothed curve for super-enhancer size density. b) Normalized ChIP-seq profiles at exemplar super-enhancers in B6. +/+ mtec hi. Numbers to the right indicate the ranges of normalized tag densities., super-enhancer. Data are representative of two independent experiments. Data for one of the ChIP-seq experiments in (a, b) came from (ref.22).
Supplementary Figure 3 Super-enhancer repertoire of HEK293T cells. a) Hockey plot for delineation of super-enhancers in HEK293T cells with ChIP-seq experiment reported in (ref. 24), super-enhancer; CE, conventional enhancer. b) Heatmaps of tag density for the indicated proteins 500kb up- or down-stream of -delimited super-enhancers in HEK293T cells, and H3K4me1 data came from experiments reported in (ref. 24) while, RNA-PolII and data were reported in (ref. 14).
Supplementary Figure 4 shrna knockdown of -partners to reveal the sequence of -partner associations in HEK293T cells. Primary data supporting Fig. 3e. IP, immunoprecipitation. Representative immunoblots from two independent experiments.
FLAG FLAG + EtBr FLAG FLAG + EtBr LacZ-2 TOP2B -1 TOP2B -3 TOP2B -4 LacZ-2 TOP2B -1 TOP2B -2 TOP2B -3 TOP2B -4 LacZ-2-1 -2-3 -4 LacZ-2-1 -2-3 -4 a b Input FLAG () IP Input FLAG () IP shrna TOP2B -2 shrna Blotting Ab FLAG () DNA-PKcs Blotting Ab FLAG () DNA-PKcs Ku80 Ku80 PARP-1 PARP-1 BRD4 BRD4 CDK9 (ptefb) CDK9 (ptefb) TOP2B SPT5 (DSIF) SPT5 (DSIF) RNA-PolII RNA-PolII DDX5 DDX5 SFRS3 SFRS3 c Input IP Post pre-clearing supernatant IP with anti-flag d Input IP Blotting Ab Preclearing Ab FLAG () DNA-PKcs Ku80 Blotting Ab IP Ab FLAG () DNA-PKcs PARP-1 Ku80 BRD4 CDK9 (ptefb) TOP2B SPT5 (DSIF) RNA-PolII DDX5 SFRS3 PARP-1 BRD4 CDK9 (ptefb) TOP2B SPT5 (DSIF) RNA-PolII Supplementary Figure 5 A B C D E F G H knockdown, pre-clearing experiments to confirm its primacy and DNA-dependent assembly of complexes in HEK293T cells. a, b) Primary data supporting Fig. 4b,c. c) Primary data in support of Fig. 4e. d) Loss of ability of to interact with its partners in the presence of ethidium bromide (EtBr) (100mg/ml). IP, immunoprecipitation. Representative immunoblots from two independent experiments.
a b [0-66] [0-125] [0-66] [0-125] [0-16] [0-16] [0-13] [0-13] [0-26] [0-26] Grap Slc5a10 Zkscan17 Olfr225 Mprip Pld6 Flcn [0-32] [0-63] [0-32] [0-63] [0-17] [0-17] [0-13] [0-13] [0-32] [0-32] Il13 Rad50 Fstl4 [0-28] [0-79] [0-28] [0-79] [0-23] [0-23] [0-14] [0-14] [0-59] [0-59] Syn3 Syn3 Aldh1l2 A230046K03Rik Appl2 Supplementary Figure 6 Differential distribution of and at super-enhancers in mtecs. a, b) Normalized ChIP-seq profiles of indicated proteins at exemplar super-enhancers (a) or non-super-enhancer regions (b) in B6. +/+ mtec hi to emphasize the lack of enrichment of at super-enhancers. Numbers to the right indicate the ranges of normalized tag densities., super-enhancer. Data are representative of two independent experiments. Data for one of the ChIP-seq experiments in (a, b) came from (ref. 22).
Supplementary Figure 7 Lack of thymic stromal cell and thymocyte perturbations after inhibition of topoisomerases. a, b) Representative cytofluorimetric plots and summary quantitative data for mtec hi (a) and various thymocyte compartments (b) of B6. +/+ mice treated with the indicated topoisomerase inhibitor/s or just vehicle (DMSO) every day for 3 days. Data are representative of three independent experiments with similar results (error bars, mean ± s.e.m. from n=3 measurements pooled from three experiments).
Supplementary Figure 8 Effect of topoisomerase inhibitors on mtec gene expression and autoimmunity. a, b) Representative cytofluorimetric plots gated on CD45 - Ly51 lo MHCII hi mtecs (b) and summary quantitative data (a, b) for mtec expression of (a) or the topoisomerases (b) in B6. +/+ mice treated with the indicated topoisomerase inhibitor/s or just vehicle (DMSO) every day for 3 days. MFI, mean fluorescence intensity. c) As per Fig. 7a, except total RNA was isolated from mtecs after three weeks of treatment of mice with topoisomerase inhibitors. Gene-expression analyses were performed by quantitative PCR using SYBR Green. d) As per Fig. 6a, except 4-week-old B6. -/- mice were intra-peritoneally injected with topotecan (n=3), etoposide (n=2), both drugs (n=2) or just vehicle (DMSO) (n=3). Orange: transcripts increased >2-fold in vehicle-treated +/+ vs -/- mice; green: transcripts decreased >2-fold. Numbers refer to transcripts up- (right) or down- (left) regulated by the indicated inhibitors (etoposide, P = 1.6 x 10-45 ; topotecan, P = 4 x 10-58 ; etoposide + topotecan, P = 9 x 10-101, P values for -induced genes from χ2 test). e) Organ histology scores at 8-12 weeks of age for NOD/LtJ pups ( -/- ) intra-peritoneally injected with topotecan, etoposide or just vehicle on the 2nd, 4th and 8th day after birth (6-7 mice per group). Scores reflect the scale described in Methods. *P < 0.05 (unpaired Student s t-test). Data are representative of at least two (d, e) or three independent experiments (a-c) (error bars, mean ± s.e.m. from n = 3 (a-c) or n 6 (e) measurements).
Supplementary Figure 9 A simplified model of s mechanism of action. A detailed scenario is presented in the Discussion section of the text. Not all partners are depicted here, just those highlighted in the text. 1) At the super-enhancer, and interact, resulting in stabilization of DNA DSBs, mobilization of the DNA-damage response (e.g., DNA-PKcs, Ku80, PARP-1) and recruitment of general transcription factors (e.g. CBP, BRD4). 2) At the TSS or just downstream of it, additional DSBs occur due to contortion induced by paused RNA-PolII. The superenhancer delivers its abundant -containing complexes to the TSS, which overcomes pausing and promotes elongation. 3) Effective elongation necessitates relief of torsional stress up- and down-stream of the advancing RNA-PolII as well as eviction of interfering nucleosomes, likely performed by the histone eviction complex (including TOP2, DNA- PKcs, Ku80, PARP-1. etc). 4) An as-yet poorly defined -containing complex promotes pre-mrna processing. Ac = an acetylated residue; me1 = addition of a single methyl group; me3 = a trimethylated site; P = a phosphorylation site; TSS = transcriptional start-site; ptefb is depicted as having two subunits, CycT1 and CDK9; bold red stretch = super-enhancer; bold black stretch = TSS; bold green = emerging pre-mrna transcript.