b PolyA RNA-seq c RNA-seq read distribution FPKM
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1 a Repliate 2 (FPKM) Poly+ RN-seq R2 = Repliate 1 (FPKM) Repliate 2 (FPKM) Poly RN-seq R2 = Repliate 1 (FPKM) RN-seq rea istriution Intron 12% 14% Intergeni Poly+ 77% Exon Intron Intergeni 27% 36% Poly 38% Exon anonial histones Hist3h3 Hist3h2 Hist3h2a Hist3h2a Hist2h4 Hist2h2e Hist2h2a Hist2h2a Hist2h2aa3 Hist1h4m Hist1h4 Hist1h3 Hist1h2q Hist1h2o Hist1h2l Hist1h2h Hist1h2f Hist1h2 Hist1h2l1 Hist1h2 Hist1h2a Hist1h2ao Hist1h2ak Hist1h2ail1 Hist1h2ail Hist1h2ail Hist1h2ai Hist1h2ah Hist1h2ah Hist1h2af Hist1h2a Hist1h2aa Hist1h1 Hist1h1 Hist1h1a Poly+ Poly FPKM Supplementary Figure 1. Poly+ an Poly RN-seq valiation. a, orrelation etween iniviual iologial repliates for Poly+ RN-seq (linear regression, P < 1)., orrelation etween iniviual iologial repliates for Poly RN-seq (linear regression, P < 1)., Distriution of mappe sequene reas in Poly+ (top) an Poly (ottom) RN-seq. Perentages sum to greater than ue to rea overhang etween features., Expression estimates from Poly+ an Poly RN-seq for anonial histone transripts, whih are known to lak polyaenylation signals. Reas mapping to these transripts were more aunant in the Poly lirary for all histone genes, iniating speifi seletion of Poly transripts in the Poly RN-seq. Data are expresse as mean ± s.e.m.
2 a MBD-seq Extrat genomi DN, fragment Forwar reas m TSS Reverse reas - alignment to annotate genome - ioinformatis - Inuate with MBD2, apture with magneti eas T T - lirary onstrution - next generation sequening - MBD2 Biotin Elute methylate DN Streptaviin Magneti ea ene methylation (RPM) Repliate n ou - n MBD-seq (promoter) R2 = ene methylation (RPM) Repliate 1 BD M MBD-seq (gene oy) 1-1 ap MBD-apture Promoter methylation (RPM) Repliate 2 m DN/non-m DN 1-2 R2 = Promoter methylation (RPM) Repliate 1 e ene Rattus norvegius (Rn5 assemly) hr4: pr19 rel2 kn1 pol1 Dx47 m Repliate 1 Input Repliate 2 2k Supplementary Figure 2. MBD-seq pipeline an valiation. a, MBD-seq workflow., Valiation of methylate DN apture with MBD-IP. Inuation with MBD2 resulte in roust enrihment of methylate DN in IP sample an epletion from unoun fration (n = 4 per group; 2-taile Mann-Whitney test; P = 286). Data are expresse as mean ± s.e.m., orrelation of gene oy methylation levels etween iniviual iologial repliates in MBD-seq (linear regression, P < 1 for eah omparison)., orrelation of promoter methylation levels etween iniviual iologial repliates in MBD-seq. e, Representative genomi lous highlights DN methylation patterns surrouning gene oies an lak of efine peaks in input (non-ip) sample.
3 m - input k p islan Start En Intergeni ll islans Intrageni Promoter TSS +1k ll p Islans Intergeni Intrageni Promoter Overlapping TSS - DN methylation (m - input) m - input Promoter m (-2.5 to 2.5k from TSS) Non-I p < 1 p < 1 I ern quartile m - input Intrageni pi m p < 1 ern quartile Supplementary Figure 3. enome-wie DN methylation y p islan status. a, Meta-feature profile of DN methylation at p islans, stratifie y genomi loation. TSS-spanning p islans are hypomethylate, whereas intergeni p islans are hypermethylate. Data represents average methylation aross ategories, aligne to p islan start an en loations., omparison of p islan methylation y genomi feature (n = per group; one-way NOV, F(4,35696) = 176.2; P < 1, Tukey s post-ho test for iniviual omparisons)., Promoter DN methylation aoring to ern rank, stratifie y presene (re points) or asene (orange points) of p islan (., DN methylation at intrageni p islans, y ern rank of orresponing gene. ern levels are signifiantly orrelate with methylation of intrageni p islans.
4 RNPII inhiitor DRB (4hr treatment) RNPIII inhiitor ML-6218 (4hr treatment) Fos mrn tf4 mrn Fos ern * tf4 ern Fos mrn tf4 mrn * Fos ern tf4 ern RNP3 hip Fos Promoter Kl Supplementary Figure 4. Fos an tf4 ern are ifferentially regulate y RN polymerases. a, Treatment with the RN polymerase II-epenent transriptional inhiitor 5,6-ihloro-1-eta-D-riofuranosylenzimiazole (DRB) loke Fos mrn an ern proution. DRB treatment ha no effet on tf4 ern ut erease tf4 mrn in a ose-epenent manner., Treatment with the RN polymerase III inhiitor ML-6218 ha no effet on Fos or tf4 mrn ut erease Fos an tf4 ern (n = per group for DRB experiments an per group for ML-6218 experiments; one-way NOV, Tukey s post-ho test for multiple omparisons)., fter Kl inution, RN polymerase III ining inreases at the Fos promoter (n = 2 per group; Stuent s t-test versus vehile, P = 396). ll ata are expresse as mean ± s.e.m. Iniviual omparisons, *P < 5, P < 1, *P < 1, an P <1.
5 ern-1 Sequene 5 -y Base omposition :3 :9 :6 :7 /:2 Preite seonary struture 1 proaility omplexes Proe Free proe +DNMT3a-D ern-1 ern-2 Sequene 5 -y Base omposition :5 :6 :7 :7 /:3 Preite seonary struture 1 proaility omplexes Proe Free proe +DNMT3a-D ern-2 Free proe Ig DNMT3a-D ern-1 poly I- omplexes Proe mutant ern-1 Sequene 5 -y Base omposition :3 :9 :6 :7 /:4 Preite seonary struture 1 proaility omplexes Proe Free proe +DNMT3a-D mutant ern-1 mutant ern-2 Sequene 5 -y Base omposition :5 :6 :7 :7 /:3 Preite seonary struture 1 proaility omplexes Proe Free proe +DNMT3a-D mutant ern-2 Free proe Ig DNMT3a-D ern-2 poly I- omplexes Proe Supplementary Figure 5. ern interations with DNMT3a are not regulate y stem-loop seonary struture an are maintaine in the presene of a non-speifi ompetitor. a, Base sequene, omposition, an preite seonary struture for syntheti ern-1 an mutant ern-1 proes. Mutant ern-1 possesses iential ase omposition ut laks seonary struture. DNMT3a-D ining was not impaire y removal of seonary struture., Base sequene, omposition, an preite seonary struture for syntheti ern-2 an mutant ern-2 proes. Mutant ern-2 possesses iential ase omposition ut laks seonary struture. DNMT3a-D ining is not altere in mutant ern-2., ern-1 an ern-2 proes (1nM) o not in to Ig (.2μM). Inreasing amounts of the non-speifi ompetitor poly I- (1nM-nM) oes not aolish Fos ern ining to DNMT3a-D.
6 Fos gene MeDIP DN methylation (fol versus vehile) Enhaner Promoter ene oy ile mrn SO-2 ern SO-1 * Fos mrn SO * Fos ern SO-1 treatment Fos ern SO SO treatment (ays) * n.s mrn ern Supplementary Figure 6. SO meiate knokown of Fos mrn an ern in ortial ultures. a, Fos ern knokown resulte in inrease enhaner, promoter, an gene oy methylation (n = 4 per group; two-way NOV versus vehile, Tukey s post-ho test for multiple omparisons)., SO meiate ern knokown persiste for up to 7 ays, an resulte in a progressive eline in Fos mrn expression (n = 6 per group, two-way NOV, Siak s post-ho test for multiple omparisons). Data are expresse as mean ± s.e.m. Iniviual omparisons, *P < 5, P < 1, *P < 1, an P <1.
7 1 2 juste 3 ern 3' ern aniate genes (1,771) quantifiation 5' gene X Poly+ RPM 2k 3' Poly- RPM 3' ern laking genes (6,948) Poly- RPM juste 3' ern inex = Poly+ RPM ,719 juste 3 ern rank Poly+ (FPKM) mrn p < 1 m - input juste 3 ern inex Promoter (-2.5 to 2.5k) juste 3 ern quartile (3 ern aniates only) p < 1 e tf4 ria2 Fos Poly- > Poly+ Poly- < Poly+ Promoter m perentile Promoter m - input No 3'eRN 3'eRN Density (a.u.) Min Max juste 3 ern perentile Supplementary Figure 7. lternative analysis onfirms genome-wie relationship etween ern an promoter DN methylation. a, lternative ern quantifiation ajusts ern levels for Poly+ expression for the same lous, removing potential ontamination from overlapping genes or inorret gene annotations., lternative analysis reveale at least 1,771 genes with 3 Poly- RPM greater than 3 Poly+ RPM., This group of genes exhiite less promoter methylation than genes laking 3 ern proution. -e, 3 ern status traks mrn an DN methylation.
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