Supplementary Figure 1a Hours: E-cadherin TGF-β On TGF-β Off 0 12 24 36 48 24 48 72 Vimentin βactin Fig. S1a. Treatment of AML12 cells with TGF-β induces EMT. Treatment of AML12 cells with TGF-β results in reversible loss of E-cadherin and gain of vimentin protein expression, as shown by western blot, consistent with induction of EMT. TGF-β On refers to the indicated timepoints where cells are treated with TGF-β. TGF-β Off refers to the indicated timepoints after which TGF-β was removed from the media, which allows cells to terminate EMT and redifferentiate into hepatocytes.
Supplementary Figure 1b E-cadherin TGF-β 40X 40X Fig. S1b. Loss of E-cadherin expression during EMT by immunostaining. Immunohistochemistry for E-cadherin after 36hrs of TGF-β stimulation shows loss of E-cadherin staining, consistent with induction of EMT.
Supplementary Figure 1c 0hr 20X 36hr Serum 20X 36hr Vehicle 20X 36hr TGF-β, 20X Fig. S1c. Immunostains for Ki67 for the indicated conditions reveal that AML12 cells are not entering the cell cycle during experiments. The Ki67 index was approximately 20% for serum controls whereas several fields had to be scanned to find positive nuclei for other conditions (Ki67 index << 1%).
Supplementary Figure S1d 36hrs serum 20X 36hrs vehicle 20X 36hrs TGF-β 20X Fig. S1d. AML12 cells were growth arrested, treated with BrDU and stimulated as indicated. Serum stimulated cells displayed a BrDU index of approximately 50% whereas vehicle and TGF-β treated cells had approximate indices of 0.5%, indicating that only a small % of cells proliferated during the 36hr treatments.
Supplementary Figure 2A
Supplementary Figure S2b
Fig. S2. DNA methylation does not change during EMT. Representative plots of CHARM data from differentiated AML12 cells and those treated with TGF-β. The degree of DNA methylation is indicated by p (percent methylation) value versus genomic location, where the curve represents averaged smoothed p values. The location of CpG dinucleotide (black tick marks on x axis), CpG density (smoothed black line) calculated across the region using a standard density estimator, location of CpG islands (orange line), as well as gene annotation indicating the transcript (thin outer gray line), coding region (thin inner gray line), exons (filled gray box) and gene transcription directionality on the y axis (sense marked as +, antisense as -) are also shown. Examples are shown for Rab30 and Rplp1.
Supplementary Figure 3a Zero 36hrs H3K9Me2 IF TGF-β Hoechst IF counterstain TGF-β H3K9Me2 IHC TGF-β Fig. S3a. Loss of H3K9Me2 during EMT by immunostaining. Immunofluorescence (top, with Hoeschst counterstain control, middle) and immunohistochemical (bottom) staining shows reduction in nuclear H3K9Me2 during EMT (36hrs TGF-β treatment).
Supplementary Figure 3b Zero 36hrs E-cadherin Ecad+K9Me2 merge E-cadherin Ecad+K9Me2 merge TGF-β Hoechst H3K9Me2 Hoechst H3K9Me2 Fig. S3b. Loss of H3K9Me2 and E-cadherin co-localize during EMT by immunostaining. Immunofluorescence shows reduction in nuclear H3K9Me2 and membranous E-cadherin in the same cells during EMT (36hrs TGF-β treatment). Hoechst DNA counterstain stains nuclei strongly in under both conditions.
Supplementary Figure 4 Vehicle On Vehicle Off Hours : 0 12 24 36 48 24 48 72 H3K9Me2 H3K36Me3 H3K4Me3 H3K9Ac Fig. S4. Chromatin modifications do not show changes similar to EMT when treated with vehicle alone. Western blots for indicated histone modifications from AML12 cells treated with vehicle for the indicated times (Vehicle On) and for the indicated times after vehicle was removed from the media (Vehicle Off). There is no loss of H3K9Me2 or gain of H3K36/K4Me3 in response to vehicle control.
Supplementary Figure 5 Zero 36hrs K36Me3 IF TGF-β Hoechst IF Counterstain K36Me3 IHC TGF-β TGF-β Fig. S5. Gain of H3K36Me3 during EMT by immunostaining. Immunofluorescence (top) with Hoechst counterstain (middle) and immunohistochemistry (bottom) show nuclear increases in H3K36Me3 in response to 36hrs of TGF-β stimulation.
Supplementary Figure 6 Hours : 0 12 24 36 48 24 48 72 H3K9Ac Total H3 TGF-β On TGF-β Off Fig. S6. H3K9 acetylation (H3K9Ac) does not increase during EMT. Western blots for H3K9Ac and total H3 in response to TGF-β stimulation for the indicated times, as in other experiments. H3K9Ac does not increase during TGF-β treatment (TGF-β On) despite loss of H3K9me2 (see Fig. 1B). Total H3 levels remain relatively constant throughout the TGF-β treatment.
Supplementary Figure 7 Fig. S7. Reduction of H3K9Me2 occurs specifically in LOCKs. Plotted is the amount of H3K9Me2 reduction between differentiated (no TGF-β) and EMT (+TGF-β) AML12 cells, shown for both LOCK and non-lock regions on our ChIP-chip arrays. LOCKs show much more H3K9Me2 reduction than non-locks, p=0.
Supplementary Figure 8a Fig. S8a. Replicate ChIP-chip experiments show loss of H3K9Me2 within LOCKs. ChIP-chip results over a representative region of chromosome 11 is shown. Both replicates show decrease in H3K9Me2 within this LOCK during EMT (36hrs TGF-β treatment) and gray bars show that LOCKs are detected in similar locations.
Supplementary Figure S8b Fig. S8b. Replicate ChIP-chip experiments show loss of H3K9Me2 within LOCKs. ChIP-chip results over a representative region of chromosome 10 is shown. Both replicates show decrease in H3K9Me2 within this LOCK during EMT (36hrs TGF-β treatment) and gray bars show that LOCKs are detected in similar locations.
Supplementary Figure 8c 0hrs 36hrs Off Lsd1 Ephb2 C1q Epha8
Fig. S8c. Validation of H3K9Me2 arrays by ChIP-qPCR. 30 PCR primers are spaced across a 500kb locus on chromosome 4. Off depicts removal of TGF-β for 72hrs, where EMT has reversed and cells have re-differentiated. Like the arrays, there is loss of H3K9Me2 during EMT (36hrs TGF-β). PCR also shows that this effect reverses upon removing TGF-β from the media. Error bars are S.E.M., n=2.
Supplementary Figure 9 H3K4Me3 H3K36Me3 0hrs 36hrs H3K4Me3 H3K36Me3 Lsd1 Ephb2 C1q Epha8
Fig. S9. Validation of ChIP-chip data by ChIP-qPCR for H3K4Me3 and H3K36Me3. Array results are shown in the top panel and PCR in the bottom panel, as in Fig. S8C. PCR confirms gain of H3K4Me3 across the LOCK during EMT (36hrs TGF-β treatment). Also highlighted by the PCR results are the appearance of short, sharp peaks over the transcription start site and within one area of the LSD1 gene body, one of the few regions of the genome where new peaks (rather than broad enrichments) appeared during EMT. H3K36Me3 is enriched at the LOCK boundaries, including the LSD1 and Epha8 genes. Error bars represent S.E.M., n=2.
Supplemental Table 1: Summary of Mass Spectrometry Results of LSD1 IP. Data was analyzed with Scaffold 3 software. Peptide hits fell within 75-99% confidence at the correct molecular weight. Veh=Vehicle. TGF=TGF-β. Both Enriched Vehicle Enriched TGF-β Enriched Protein #Peptides Veh/TGF Protein #Peptides Veh/TGF Fold Enriched Protein #Peptides Veh/TGF Fold Enriched BHC80 22/15 MYST4 15/3 5 DDX5 4/40 10 CTBP1 19/25 CoREST 8/1 7 DDX17 1/4 10 CTBP2 3/5 REST 3/0 10 ATP5A 0/10 99 HMG20A 9/5 STAT3 8/1 10 ATP5B 0/17 120 HMG20B 14/22 UBXD2 5/1 5 CTNNA 0/11 57 Hist. H1 14/22 WDR87 6/0 70 CTNNB 0/6 38 PLOD1 12/7 TAX1BP1 5/1 5 CTNND 0/16 89 PLOD3 5/4 MED20 4/0 59 CTNNG 0/5 23 SAP18 3/3 SETD2 3/0 33 ARF4 0/6 99 LSD1 7/11 MLH1 4/0 49 FUS/TLS 2/5 31 FANCM 4/0 48 KRAS 0/5 110 RAC1 0/4 82 PKP1 0/4 13 PABPC1 0/5 39 LMNA 0/4 13 PARP1 0/4 28
Supplemental Table 2: GO analysis of genes enriched for H3K36Me3 during EMT. Term Count % P-value phosphoprotein 745 45.2 2.20E-27 cytoplasm 380 23.0 2.23E-15 actin binding 67 4.0 3.42E-14 cytoskeletal protein binding 82 4.9 4.30E-13 actin cytoskeleton 47 2.8 4.31E-10 focal adhesion 22 1.3 2.34E-09 cytoskeleton 95 5.7 2.52E-09 nucleoside-triphosphatase regulator activity 66 4.0 6.80E-09 GTPase regulator activity 65 3.9 8.56E-09 cell-substrate adherens junction 22 1.3 9.65E-09 Pleckstrin homology-type 56 3.4 1.08E-08 intracellular non-membrane-bounded organelle 219 13.3 5.47E-07 anchoring junction 29 1.7 8.27E-07 basolateral plasma membrane 31 1.8 1.55E-06 regulation of Ras protein signal transduction 37 2.2 2.81E-06 cell projection 81 4.9 3.79E-06 PH domain 50 3.0 4.13E-06 acetylation 261 15.8 4.62E-06 actomyosin 12 0.7 5.09E-06 cell junction 69 4.1 5.48E-06 actin-filament process 35 2.1 1.02E-05 compositionally biased region:pro-rich 102 6.1 1.20E-05
cell leading edge 25 1.5 1.48E-05 phospholipid binding 26 1.5 1.65E-05 cytoplasmic membrane-bounded vesicle 61 3.7 1.85E-05 alternative splicing 458 27.8 2.16E-05 lamellipodium 17 1.0 4.50E-05 stress fiber 10 0.6 5.51E-05 cytoskeleton organization 51 3.1 7.99E-05 enzyme binding 38 2.3 9.49E-05
Supplemental Table 3: Characteristics of K4Me3 LOCKs in cells undergoing EMT. The term new refers to regions where new enrichments are observed during EMT. The term all refers to all sites that are enriched equally in both differentiated and EMT cells. P-values are based on 1000 permutations. Characteristics of K4Me3 LOCKs P-value 181/856 (21.1%) LOCKs converted to K4Me3 LOCKs <.001 190/219 (86.8%) K4Me3 enriched regions within LOCKs <.001 62/190 (32.6%) K4Me3 LOCKs flanked by new K36Me3 <.001 145/190 (76.3%) K4Me3 LOCKs flanked by all K36Me3 <.001
Supplemental Table 4: GO analysis of genes within K4Me3 LOCKs during EMT Term Count % P-value Immunoglobulin subtype 22 11.0 6.43E-10 glycoprotein 87 43.6 1.57E-9 plasma membrane 70 35.1 1.01E-7 Pregnancy(-specific glycoprotein) 6 3.0 9.58E-7 membrane 107 53.6 1.26E-6 substrate specific channel activity 18 9.0 1.32E-6 topological domain:extracellular 57 28.6 1.59E-6 ion channel activity 17 8.5 4.05E-6 topological domain:cytoplasmic 66 33.1 5.11E-6 signal peptide 68 34.1 1.08E-5 disulfide bond 57 28.6 1.61E-5 region of interest:rcl 5 2.5 2.24E-5 transmembrane region 84 42.1 4.92E-5 voltage-gated channel 10 5.0 6.84E-5 Collagen triple helix repeat 8 4.0 7.90E-5 developmental protein 25 12.5 1.60E-4 Tyrosine protein kinase 8 4.0 3.15E-4 gap junction protein 4 2.0 3.76E-4 skeletal system development 13 6.5 4.39E-4 serpin 6 3.0 4.77E-4
Supplementary Table 5. PCR primers. qrt-pcr primers Luz F LuzR Lsd1F Lsd1R ZbtF ZbtR XrccF XrccR TdrF TdrR TmemF TmemR AdssF AdssR ChstF ChstR PsapF PsapR SlcF SlcR UncF UncR EphbF EphbR Eph8F Eph8R CdhF CdhR TdrF TdrR KifF KifR TGT CAA GAA AGG GGC CAC GCA CAG C GTT AGT GAG TTC TGC CAT GTC TAC AGC CAC GTT GGC AGT ACA ACT GCT AGC TCA ACA GAG CAC CAT GGA CTG TAG C TGC AGA GAA AGT GTC TCA GAT TCA ACC CAG GAA CTC CAC AAT AGG TCT GAG C CCC ATG AGG ACG ATG TTA CC TCA GAA TCA GGG TTC CTT GG GAA CTG CAA GGC TGT TCT CC GGT CTC TCT CAG GAG CAT GG TGG GAT GCC TAG ACT TGT CC CCT GTA GCA AGC TGG AAA GG CCT GAT GAT CCT CCG ATA CG TGC AGG ATC TCC TGG TTA GC TAC AAG CGC TGT CTT TGT GC AGA ACA GAA TGC ACC CAT CC GTG CCT CCT CAG AAG AAT GG TGC TTC TGG TAA GGG TCT GG CAC ATC CTT GCA CAT CTT GG GGC TCT GGA GTG TTC TCT GG CCA GAC TCC TAC CCA TCA GC GTG TGA CGT GGT CAT TCT GG GGA CTA TGG CGG CTG TAT GT GCA CAT CCA CTT CTT CAG CA CCT GTG AGC TGG GCT TCT AC TCA GGT TCA CTG GTG CTG AG ACA GGC AAG ATC CGT ACC AC GTG CCC TCA AGA ATC TCA GC TCA ACC AAC ATC GCA GAG AG AAC GTG GTC AGG GAT AGC AC CGT GTA CCA GTA CCG TGT GG GGT GAG TCT GTG GTC CCT GT qpcr primers for ChIP (mouse chromosome 4) 1F 1R 2F 2R 3F 3R GGA AGG TGA TGT CCA CAT GAG TCT GG CGG GAA CAG GAA TCA AAC AGA CTT ACG GTA TAC ACT CTA GAT TCA TTT CTA ATC TCG AGC CTA TTT GGG GAA AGA CCA CAA GC GCA GCC TAG GCA TGT CTG ACC AAG G CCA TCA TGT GCT TTA GCT CTG TGT GC
4F 4R 5F 5R 6F 6R 7F 7R 8F 8R 9F 9R 10F 10R 11F 11R 12F 12R 13F 13R 14F 14R 15F 15R 16F 16R 17F 17R 18F 18R 19F 19R 20F 20R 21F 21R 22F 22R 23F 23R 24F 24R 25F 25R 26F CCT GAT GCC CAG GAA CAC AAA CTG G CTT AGC AGG TCA TTG TGA TCC TTC C TCT AGC TCC ACA TCC TTG CTT CTA GC TGG TTG TGA GCC ACC ATG TGG TTG C AGC ATA GGA ATC TGA GAT CTT ACG AGC GCT TAA ACA GTG CTT GAA CTT GTG AAC C GAT GGG TCA TCA CAT AGG TAC TTG C TAG AGT CTG GTA GGG CCT GAA AGT GC CCA GAT CTG GCC AAG CCA TGC TTG C TGT TGG GAT ATA GGA GTG CAG AGG GGC CAG CTC AAC CTT CCC AGA AGG GGG ACA GGC AAG AGA CGT TCG TTC C AGT TCA TAC CAG TTC CAG AGC AGA AGG GAT TCC TAA GTG GCT CCT CTG GAT CC CCT TGG GCG TCC AGT AGA TGC TGG GAC TTA GAG AAT CTC AGA AGC TAC AGC GTT TGT TGA GAC CCG AAG CTA CAG G AAT AGT CTA TGA ATC TGT GGA CAG TGG GGT CAG TGT TAG CTT TGC CTT GAT GG TAC TCA AGG TCA CAC AGC TAA GAT ACC AGC CAT GCT CTT TCT TGG ACT CAT GG AAG TGA GGG GTG AAC TGA CTG TTG G GGC CTG AGG CTA GAG TCA TCA CAG G CTA AGC ATA TGC AAC AAG TCA GAT GTG C ACC GAC CGA CTG TCT CAG TCT TGG GTT TCA GGA TGG ATG GGC GTC AGG AGA GAG GTG GTA GAG ACG AGT TGG CAC GGA GCC AGA CTT CAG ACG TGC CCA CGA GTC TAC TCA TCT ACC GAT CC AGC AGT GGG CTG AGC AGT TGA CAG C CCA AGC TAC TGA AGA TGA CTC AGT GG GAG TGT GGG CAT GCA TGT GTC ATG G TAC TCT CCT GCA GAC AAG AGG ATA GC ACA GGA TTC ATC CTA TGT CTC ACA GC TTT GTA TGA GCC CCA CTA CTG ACT GG ACT TTG GGA ACA AGA ATG TTG CTG TCC TCA GTT GAA CAA CTG GCT AAT GTG ACC GTG TTT GCT TCT GAG ATG CAC AGT GC GTG TGC TGG CTC ACA CTT ACA CTC C TGT GCT GAG AAT TGA GCT CAT ACT TGC GGC AAC TCC ATG CTA GGC ACA GAG C CTC AGC AAG TCA AGG TAC TTT GTC ACC GGC TGG CCA AGG AAG GTC CAA GTG C ATG CAC AGT TTT CTT GTG TAA TCC TGC TCT GAC TGA CCA GAA ATA CGT GAA TGG
26R 27F 27R 28F 28R 29F 29R 30F 30R 31F 31R GCA GGG CTA AGT ATC AAA CCC ACT GC CAG GGG TGA TGC AGA GCC AGT CG ATT AAT CTG GCC TTG ACA CGT GAT AGC AGT CAG CAG TCA GGG CTG AAG TCC TTT AGA TGG CCT GGT CTG TCC AAC C ACC GGG ACT CAT TTA AGA GCA CTG G GCG CAT CCC TCG GAG CTG TCA CC GTG CTC TTC ACG CCT GGA GTC AGC GTA CTT AGT GAA TGC ACA CTT AGC ATCC CAA ATG TCT TGT GAT GAC AGA CTG TGC CTC TAG GAG AGA CCT GGT TCT TGA CC