V6.5 murine embryonic stem (mES) cells were grown in 2i + LIF conditions. mES cells were always grown on 0.2% gelatinized (Sigma, G1890) tissue culture plates. The media used for 2i + LIF media conditions is as follows: 967.5 mL DMEM/F12 (GIBCO 11320), 5 mL N2 supplement (GIBCO 17502048), 10 mL B27 supplement (GIBCO 17504044), 0.5mML-glutamine (GIBCO 25030), 0.5X non-essential amino acids (GIBCO 11140), 100 U/mL Penicillin-Streptomycin (GIBCO 15140), 0.1 mM b-mercaptoethanol (Sigma), 1 uM PD0325901 (Stemgent 04- 0006), 3 uM CHIR99021 (Stemgent 04-0004), and 1000 U/mL recombinant LIF (ESGRO ESG1107). For differentiation mESCs were cultured in serum media as follows: DMEM (Invitrogen, 11965-092) supplemented with 15% fetal bovine serum (Hyclone, characterized SH3007103), 100 mM nonessential amino acids (Invitrogen, 11140-050), 2 mM L-glutamine (Invitrogen, 25030-081), 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, 15140-122), and 0.1mM b-mercaptoethanol (Sigma Aldrich). HEK293T cells were purchased from ATCC (ATCC CRL-3216) and cultured in DMEM, high glucose, pyruvate (GIBCO 11995-073) with 10% fetal bovine serum (Hyclone, characterized SH3007103), 100 U/mL Penicillin-Streptomycin (GIBCO 15140), 2 mM L-glutamine (Invitrogen, 25030-081). Cells were negative for mycoplasma.

Method Details

Immunofluorescence with RNA FISH Coverslips were coated at 37°C with 5ug/mL poly-L-ornithine (Sigma-Aldrich, P4957) for 30 minutes and 5 μg/mL of Laminin (Corning, 354232) for 2 hours. Cells were plated on the pre-coated coverslips and grown for 24 hours followed by fixation using 4% paraformaldehyde, PFA, (VWR, BT140770) in PBS for 10 minutes. After washing cells three times in PBS, the coverslips were put into a humidifying chamber or stored at 4°C in PBS. Permeabilization of cells were performed using 0.5% Triton X-100 (Sigma Aldrich, X100) in PBS for 10 minutes followed by three PBS washes. Cells were blocked with 4% IgG-free Bovine Serum Albumin, BSA, (VWR, 102643-516) for 30 minutes and the indicated primary antibody (see table S2) was added at a concentration of 1:500 in PBS for 4-16 hours. Cells were washed with PBS three times followed by incubation with secondary antibody at a concentration of 1:5000 in PBS for 1 hour. After washing twice with PBS, cells were fixed using 4% paraformaldehyde, PFA, (VWR, BT140770) in PBS for 10 minutes. After two washes of PBS, Wash buffer A (20% Stellaris RNA FISH Wash Buffer A (Biosearch Technologies, Inc., SMF-WA1-60), 10% Deionized Formamide (EMD Millipore, S4117) in RNase-free water (Life Technologies, AM9932) was added to cells and incubated for 5 minutes. 12.5 μM RNA probe ( Table S4 , Stellaris) in Hybridization buffer (90% Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies, SMF-HB1-10) and 10% Deionized Formamide) was added to cells and incubated overnight at 37C. After washing with Wash buffer A for 30 minutes at 37°C, the nuclei were stained with 20 μm/mL Hoechst 33258 (Life Technologies, H3569) for 5 minutes, followed by a 5 minute wash in Wash buffer B (Biosearch Technologies, SMF-WB1-20). Cells were washed once in water followed by mounting the coverslip onto glass slides with Vectashield (VWR, 101098-042) and finally sealing the coverslip with nail polish (Electron Microscopy Science Nm, 72180). Images were acquired at an RPI Spinning Disk confocal microscope with a 100x objective using MetaMorph acquisition software and a Hammamatsu ORCA-ER CCD camera (W.M. Keck Microscopy Facility, MIT). Images were post-processed using Fiji Is Just ImageJ (FIJI).

Immunofluorescence with DNA FISH DNA FISH probes were custom designed and generated by Agilent to target Nanog and MiR290 super enhancers.

Nanog

Design Input Region – mm9

chr6 122605249 – 122705248

Design Region – mm9

chr6: 122605985-122705394

Mir290

Design Region – mm10

chr7: 3141151 – 3241381 Immunofluorescence was performed as previously described above. After incubating the cells with the secondary antibodies, cells were washed three times in PBS for 5min at RT, fixed with 4% PFA in PBS for 10min and washed three times in PBS. Cells were incubated in 70% ethanol, 85% ethanol and then 100% ethanol for 1 minute at RT. Probe hybridization mixture was made mixing 7 μL of FISH Hybridization Buffer (Agilent G9400A), 1 μL of FISH probe (see below for region) and 2 μL of water. 5 μL of mixture was added on a slide and coverslip was placed on top (cell-side toward the hybridization mixture). Coverslips were sealed using rubber cement. Once rubber cement solidified, genomic DNA and probes were denatured at 78°C for 5 minutes and slides were incubated at 16°C in the dark O/N. The coverslip was removed from the slide and incubated in pre-warmed Wash buffer 1 (Agilent, G9401A) at 73°C for 2 minutes and in Wash Buffer 2 (Agilent, G9402A) for 1 minute at RT. Slides were air dried and nuclei were stained with Hoechst in PBS for 5 minutes at RT. Coverslips were washed three times in PBS, mounted on slides using Vectashield and sealed with nail polish. Images were acquired on an RPI Spinning Disk confocal microscope with a 100x objective using MetaMorph acquisition software and a Hammamatsu ORCA-ER CCD camera (W.M. Keck Microscopy Facility, MIT). Images were post-processed using FIJI.

Tissue culture V6.5 murine embryonic stem cells (mESCs) were a gift from the Jaenisch lab. Cells were grown on 0.2% gelatinized (Sigma, G1890) tissue culture plates in 2i media (DMEM-F12 (Life Technologies, 11320082), 0.5X B27 supplement (Life Technologies, 17504044), 0.5X N2 supplement (Life Technologies, 17502048), an extra 0.5mM L-glutamine (GIBCO, 25030-081), 0.1mM b-mercaptoethanol (Sigma, M7522), 1% Penicillin Streptomycin (Life Technologies, 15140163), 0.5X nonessential amino acids (GIBCO, 11140-050), 1000 U/ml LIF (Chemico, ESG1107), 1 μM PD0325901 (Stemgent, 04-0006-10), 3 μM CHIR99021 (Stemgent, 04-0004-10)). Cells were grown at 37°C with 5% CO2 in a humidified incubator. For confocal imaging, cells were grown on glass coverslips (Carolina Biological Supply, 633029), coated with 5 μg/mL of poly-L-ornithine (Sigma Aldrich, P4957) for 30 minutes at 37°C and with 5 μg/ml of Laminin (Corning, 354232) for 2hrs-16hrs at 37°C. For passaging, cells were washed in PBS (Life Technologies, AM9625), 1000 U/mL LIF. TrypLE Express Enzyme (Life Technologies, 12604021) was used to detach cells from plates. TrypLE was quenched with FBS/LIF-media ((DMEM K/O (GIBCO, 10829-018), 1X nonessential amino acids, 1% Penicillin Streptomycin, 2mM L-Glutamine, 0.1mM b-mercaptoethanol and 15% Fetal Bovine Serum, FBS, (Sigma Aldrich, F4135)). Cells were spun at 1000rpm for 3 minutes at RT, resuspended in 2i media and 5x106 cells were plated in a 15 cm dish. For differentiation of mESCs, 6000 cells were plated per well of a 6 well tissue culture dish, or 1000 cells were plated per well of a 24 well plate with a laminin coated glass coverslip. After 24 hours, 2i media was replaced with FBS media (above) without LIF. Media was changed daily for 5 days, cells were then harvested.

Western blot Cells were lysed in Cell Lytic M (Sigma-Aldrich C2978) with protease inhibitors (Roche, 11697498001). Lysate was run on a 3%–8% Tris-acetate gel or 10% Bis-Tris gel or 3%–8% Bis-Tris gels at 80 V for ∼2 hr, followed by 120 V until dye front reached the end of the gel. Protein was then wet transferred to a 0.45 μm PVDF membrane (Millipore, IPVH00010) in ice-cold transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) at 300 mA for 2 hours at 4°C. After transfer the membrane was blocked with 5% non-fat milk in TBS for 1 hour at room temperature, shaking. Membrane was then incubated with 1:1,000 of the indicated antibody ( Table S2 ) diluted in 5% non-fat milk in TBST and incubated overnight at 4°C, with shaking. In the morning, the membrane was washed three times with TBST for 5 minutes at room temperature shaking for each wash. Membrane was incubated with 1:5,000 secondary antibodies for 1 hr at RT and washed three times in TBST for 5 minutes. Membranes were developed with ECL substrate (Thermo Scientific, 34080) and imaged using a CCD camera or exposed using film or with high sensitivity ECL.

Chromatin immunoprecipitation (ChIP) qPCR and sequencing mES were grown to 80% confluence in 2i media. 1% formaldehyde in PBS was used for crosslinking of cells for 15 minutes, followed by quenching with Glycine at a final concentration of 125mM on ice. Cells were washed with cold PBS and harvested by scraping cells in cold PBS. Collected cells were pelleted at 1000 g for 3 minutes at 4°C, flash frozen in liquid nitrogen and stored at −80°C. All buffers contained freshly prepared cOmplete protease inhibitors (Roche, 11873580001). Frozen crosslinked cells were thawed on ice and then resuspended in lysis buffer I (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors) and rotated for 10 minutes at 4°C, then spun at 1350 rcf., for 5 minutes at 4°C. The pellet was resuspended in lysis buffer II (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors) and rotated for 10 minutes at 4°C and spun at 1350 rcf. for 5 minutes at 4°C. The pellet was resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100, protease inhibitors) and then sonicated on a Misonix 3000 sonicator for 10 cycles at 30 s each on ice (18-21 W) with 60 s on ice between cycles. Sonicated lysates were cleared once by centrifugation at 16,000 rcf. for 10 minutes at 4°C. Input material was reserved and the remainder was incubated overnight at 4°C with magnetic beads bound with antibody ( Table S2 ) to enrich for DNA fragments bound by the indicated factor. Beads were washed twice with each of the following buffers: wash buffer A (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), wash buffer B (50 mM HEPES-KOH pH 7.9, 500 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), wash buffer C (20 mM Tris-HCl pH8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% Na-Deoxycholate, 0.5% IGEPAL C-630, 0.1% SDS), wash buffer D (TE with 0.2% Triton X-100), and TE buffer. DNA was eluted off the beads by incubation at 65°C for 1 hour with intermittent vortexing in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65°C. To purify eluted DNA, 200 μL TE was added and then RNA was degraded by the addition of 2.5 μL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 10 μL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 55°C for 2 hours. A phenol:chloroform:isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in 50 μL TE and used for either qPCR or sequencing. For ChIP-qPCR experiments, qPCR was performed using Power SYBR Green mix (Life Technologies #4367659) on either a QuantStudio 5 or a QuantStudio 6 System (Life Technologies).

RNA-Seq RNA-Seq was performed in the indicated cell line with the indicated treatment, and used to determine expressed genes. RNA was isolated by AllPrep Kit (QIAGEN 80204) and polyA selected libraries were prepared using the KAPA mRNA HyperPrep Kit (Kapa Biosystems KK8505) according to manufacturer’s protocol, and single-end sequenced on a Hi-seq 2500 instrument.

Protein purification cDNA encoding the genes of interest or their IDRs were cloned into a modified version of a T7 pET expression vector. The base vector was engineered to include a 5′ 6xHIS followed by either mEGFP or mCherry and a 14 amino acid linker sequence “GAPGSAGSAAGGSG.” NEBuilder® HiFi DNA Assembly Master Mix (NEB E2621S) was used to insert these sequences (generated by PCR) in-frame with the linker amino acids. Vectors expressing mEGFP or mCherry alone contain the linker sequence followed by a STOP codon. Mutant sequences were synthesized as geneblocks (IDT) and inserted into the same base vector as described above. All expression constructs were sequenced to ensure sequence identity. For protein expression, plasmids were transformed into LOBSTR cells (gift of Chessman Lab) and grown as follows. A fresh bacterial colony was inoculated into LB media containing kanamycin and chloramphenicol and grown overnight at 37°C. Cells containing the MED1-IDR constructs were diluted 1:30 in 500ml room temperature LB with freshly added kanamycin and chloramphenicol and grown 1.5 hours at 16°C. IPTG was added to 1mM and growth continued for 18 hours. Cells were collected and stored frozen at −80°C. Cells containing all other constructs were treated in a similar manner except they were grown for 5 hours at 37°C after IPTG induction. Pellets of 500ml of cMyc and Nanog cells were resuspended in 15ml of denaturing buffer (50mM Tris 7.5, 300mM NaCl, 10mM imidazole, 8M Urea) containing cOmplete protease inhibitors (Roche,11873580001) and sonicated (ten cycles of 15 s on, 60 s off). The lysates were cleared by centrifugation at 12,000 g for 30 minutes and added to 1ml of Ni-NTA agarose (Invitrogen, R901-15) that had been pre-equilibrated with 10 volumes of the same buffer. Tubes containing this agarose lysate slurry were rotated for 1.5 hours. The slurry was poured into a column, washed with 15 volumes of the lysis buffer and eluted 4 X with denaturing buffer containing 250mM imidazole. Each fraction was run on a 12% gel and proteins of the correct size were dialyzed first against buffer (50mM Tris pH 7.5, 125Mm NaCl, 1Mm DTT and 4M Urea), followed by the same buffer containing 2M Urea and lastly 2 changes of buffer with 10% Glycerol, no Urea. Any precipitate after dialysis was removed by centrifugation at 3.000rpm for 10 minutes. All other proteins were purified in a similar manner. 500ml cell pellets were resuspended in 15ml of Buffer A (50mM Tris pH7.5, 500 mM NaCl) containing 10mM imidazole and cOmplete protease inhibitors, sonicated, lysates cleared by centrifugation at 12,000 g for 30 minutes at 4°C, added to 1ml of pre-equilibrated Ni-NTA agarose, and rotated at 4°C for 1.5 hours. The slurry was poured into a column, washed with 15 volumes of Buffer A containing 10mM imidazole and protein was eluted 2 X with Buffer A containing 50mM imidazole, 2 X with Buffer A containing 100mM imidazole, and 3 X with Buffer A containing 250mM imidazole. Alternatively, the resin slurry was centrifuged at 3,000rpm for 10 minutes, washed with 15 volumes of Buffer and proteins were eluted by incubation for 10 or more minutes rotating with each of the buffers above (50mM, 100mM and 250mM imidazole) followed by centrifugation and gel analysis. Fractions containing protein of the correct size were dialyzed against two changes of buffer containing 50mM Tris 7.5, 125mM NaCl, 10% glycerol and 1mM DTT at 4°C.

In vitro droplet assay Recombinant GFP or mCherry fusion proteins were concentrated and desalted to an appropriate protein concentration and 125mM NaCl using Amicon Ultra centrifugal filters (30K MWCO, Millipore). Recombinant proteins were added to solutions at varying concentrations with indicated final salt and 10% PEG-8000 as crowding agent in Droplet Formation Buffer (50mM Tris-HCl pH 7.5, 10% glycerol, 1mM DTT). The protein solution was immediately loaded onto a homemade chamber comprising a glass slide with a coverslip attached by two parallel strips of double-sided tape. Slides were then imaged with an Andor confocal microscope with a 150x objective. Unless indicated, images presented are of droplets settled on the glass coverslip. For experiments with fluorescently labeled polypeptides, the indicated decapeptides were synthesized by the Koch Institute/MIT Biopolymers & Proteomics Core Facility with a TMR fluorescent tag. The protein of interest was added Buffer D with 125mM NaCl and 10% Peg-8000 with the indicated polypeptide and imaged as described above. For FRAP of in vitro droplets 5 pulses of laser at a 50us dwell time was applied to the droplet, and recovery was imaged on an Andor microscope every 1 s for the indicated time periods. For estrogen stimulation experiments, fresh B-Estradiol (E8875 Sigma) was reconstituted to 10mM in 100% EtOH then diluted in 125mM NaCl droplet formation buffer to 100uM. One microliter of this concentrated stock was used in a 10uL droplet formation reaction to achieve a final concentration of 10uM.

Genome editing and protein degradation The CRISPR/Cas9 system was used to genetically engineer ESC lines. Target-specific oligonucleotides were cloned into a plasmid carrying a codon-optimized version of Cas9 with GFP (gift from R. Jaenisch). The sequences of the DNA targeted (the protospacer adjacent motif is underlined) are listed in the same table. For the generation of the endogenously tagged lines, 1 million Med1-mEGFP tagged mES cells were transfected with 2.5 mg Cas9 plasmid containing the guide sequence below (pX330-GFP-Oct4) and 1.25 mg non-linearized repair plasmid 1 (pUC19-Oct4-FKBP-BFP) and 1.25 mg non-linearized repair plasmid 2 (pUC19-Oct4-FKBP-mcherry) ( Table S3 ). Cells were sorted after 48 hours for the presence of GFP. Cells were expanded for five days and then sorted again for double positive mCherry and BFP cells. Forty thousand mCherry+/BFP+ sorted cells were plated in a six-well plate in a serial dilution. The cells were grown for approximately one week in 2i medium and then individual colonies were picked using a stereoscope into a 96-well plate. Cells were expanded and genotyped by PCR, degradation was confirmed by western blot and IF. Clones with a homozygous knock-in tag were further expanded and used for experiments. A clonal homozygous knock-in line expressing FKBP tagged Oct4 was used for the degradation experiments. Cells were grown in 2i and then treated with dTAG-47 at a concentration of 100 nM for 24 hours, then harvested. Oct4 Guide sequence tgcattcaaactgaggcacc∗NGG(PAM)

GAL4 transcription assay Transcription factor constructs were assembled in a mammalian expression vector containing an SV40 promoter driving expression of a GAL4 DNA-binding domain. Wild-type and mutant activation domains of Oct4 and Gcn4 were fused to the C terminus of the DNA-binding domain by Gibson cloning (NEB 2621S), joined by the linker GAPGSAGSAAGGSG. These transcription factor constructs were transfected using Lipofectamine 3000 (Thermofisher L3000015) into HEK293T cells (ATCC CRL-3216) or V6.5 mouse embryonic stem cells, that were grown in white flat-bottom 96-well assay plates (Costar 3917). The transcription factor constructs were co-transfected with a modified version of the PGL3-Basic (Promega) vector containing five GAL4 upstream activation sites upstream of the firefly luciferase gene. Also co-transfected was pRL-SV40 (Promega), a plasmid containing the Renilla luciferase gene driven by an SV40 promoter. 24 hours after transfection, luminescence generated by each luciferase protein was measured using the Dual-glo Luciferase Assay System (Promega E2920). The data as presented has been controlled for Renilla luciferase expression.

Lac binding assay Constructs were assembled by NEB HIFI cloning in pSV2 mammalian expression vector containing an SV40 promoter driving expression of a CFP-LacI fusion protein. The activation domains and mutant activation domains of Gcn4 were fused by the c-terminus to this recombinant protein, joined by the linker sequence GAPGSAGSAAGGSG. U2OS-268 cells containing a stably integrated array of ∼51,000 Lac-repressor binding sites (a gift of the Spector laboratory) were transfected using lipofectamine 3000 (Thermofisher L3000015). 24 hours after transfection, cells were plated on fibronectin-coated glass coverslips. After 24 hours on glass coverslips, cells were fixed for immunofluorescence with a MED1 antibody ( Table S2 ) as described above and imaged, by spinning disk confocal microscopy.