Dynamic enhancer-gene body contacts during transcription elongation

Transcription

1 Dynamic enhancer-gene body contacts during transcription elongation Kiwon Lee, Chris C.-S. Hsiung,, Peng Huang, Arjun Raj, *, and Gerd A. Blobel Division of Hematology, The Children s Hospital of Philadelphia, Philadelphia, PA 94, USA. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 94, USA Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 94, USA * Correspondence: blobel@ .chop.edu Supplemental Figure Legends Figure S. LCR contacts with the end of the β-globin depend on transcription elongation. (A) Map of β-globin gene. Numbers indicate exons. Bars denote regions used for primary transcripts analysis and ChIP. (B) Levels of β-globin primary transcripts in GE cells -/+ GATA, prior to DRB treatment and following release as measured by RT-qPCR with indicated primers. (C) pan-rnapii-chip was analyzed with indicated primers. The transcriptionally inactive. y and βh promoters served as negative controls. (D) C using DNase hypersensitive site (HS) of the LCR as an anchor. Gray striped bars indicate analyzed BglII fragments. The bar graph (right panel) compares interaction frequencies between the LCR and end of β-globin gene as indicated by the line graph with an arrow. * p=.5 and ** p=.8, respectively. Data are plotted as mean ± standard deviation of three independent experiments. Figure S. Effects of DRB treatment on Kit transcription (A) Loss of Kit primary transcript detection following DRB treatment. 8S rrna was used as control. Numbers on the x-axis indicate positions of intronic primers used for RT-qPCR.

2 Results are plotted as % of control treated samples. n=. (B) Relationship between the length of primary transcripts and elongation time. (C) Elongation rates calculated per.5 min interval. The leading edge of transcription was defined at the point at which PT level exceeded % of the steady state i.e. asynchronous cells. n=. Figure S. RNA-FISH with probes against exon, end intron, and 5 end intron under DRB blocking and release condition. (A) Nascent RNAs were detected with indicated probes in asynchronous GE cells and DRB released GE cells at indicated time points. Top row: exon, middle row: 5 end intron, and bottom row: end intron. Kit PT kinetics with heat maps is shown at the bottom of the images. (B) Fraction of cells was quantified with more than one intron spot in a cell. The dashed horizontal lines indicate those of asynchronous cells. Figure S4. Distribution of SPT5 along the Kit gene during transcriptional elongation. SPT5-ChIP was performed under the same condition as RNAPII. n=; error bars denote standard deviation of the levels of fraction of input. IgG was used as a negative control. Heat maps on the each x-axis represent the intensity of their values (Red: high and white: low). Figure S5. An elongation factor-containing enhancer/promoter complex persists stably throughout transcription elongation. (A) C measuring interaction frequencies between enhancer (-4) and promoter proximal region (+.) at indicated elongation time points. A segment at -54. served as negative control. (B) Occupancies of elongation factor SPT5 and RNAPII at enhancer (-4) and promoter proximal region (+5), respectively.

3 Figure S6. Heat maps were generated by calculating the average normalized signal intensities of two adjacent data points and converted into color scale with minimum being white (RGB: 55, 55, 55), midpoint yellow at 4% (RGB: 55, 5, ), and maximum red (RGB: 55,, ). Aligned display of heat maps of Kit locus (A, relating to Figs., and ) and CD4 locus (B, relating to Fig.4). (C) Heat maps of the Kit locus for experiment in Fig.S8 and 9. Figure S. C quality controls. (A) Preparation of C BAC standard for the Kit locus. A Kitcontaining BAC undigested (lane ) or cleaved with BglII (lane ) (left panel), and re-ligated (lane 4). The re-ligated material is expected to generate random ligation products and served as a standard for the C assay. (B) Validation of C primers using serial dilutions of BAC material were analyzed by agarose gel electrophoresis. M: bp ladder. (C) Linearity of C primers. R² was determined measuring the correlation between expected and observed signal at indications dilutions. (D) Digestion efficiencies of DRB min as measured by qpcr using primer pairs (listed in the Supplement) to amplify genomic regions with or without BglII digestion sites. n=4 with standard deviations. Figure S8. C at Kit locus with different crosslinking condition (% formaldehyde for 5min). Top: map of Kit locus; E in white circle; enhancer, P in black circle; promoter. Gray striped bars indicate analyzed BglII fragments and a dark gray bar indicates the anchor fragment (-4; enhancer) for C. Heat maps on the each x-axis represent the intensity of their values representing progression of transcriptional elongation (Red: high and white: low). N=, error bars represent standard deviation. *; p <.5

4 Figure S9. Direct comparison between RNAPII occupancies and enhancer contacts at Kit locus during transcriptional elongation. Top: experimental scheme and map of Kit locus; cells were crosslinked in % formaldehyde for min and divided for RNAPII-ChIP and C assays for each time point after DRB release. E in white circle; enhancer, P in black circle; promoter. ChIP and C results were normalized to those of promoter region of Kit. The green line indicates RNAPII (N) ChIP and the dark blue line indicates C signal. Gray striped bars indicate analyzed BglII fragments and a dark gray bar is a position of anchor for C. Figure S. Interaction frequency of neighboring gene, IFT5, with CD4 enhancer (A) Tracks of ChIP-seq and DNaseI hypersensitive sites DHS, and HK4me (Wu et al. ), and RNAPII (Hsiung et al. unpublished) on the PSU Genome Brower on Mouse July (NCBI/mm9) Assembly. The CD4 enhancer (-8kb) is defined by capture-c and variety of enhancer marks (Hughes et al. 4). Top: map of CD4 and IFT5 locus, black bars indicate BglII fragments that were examined. Blue vertical bar; an anchor site (-8kb; anchor symbol on top), Red vertical bar; IFT5 promoter region. (B) The bar graph shows relative interaction frequencies compared to that of CD4 promoter with enhancer (-8kb). *; p <. Figure S. Models of the gene topology during transcription elongation. Model A represents the conventional view of transcription elongation in which RNAPII separates from the enhancer-promoter complex. Model B, which is favored by the present study, suggests that the gene body and enhancer-promoter complex are mobile relative to each other. In this manner both the enhancer and promoter dynamically interact with the transcribed portion of the gene. One corollary of model B is that RNAPII always remains in contact with both the enhancer 4

5 and promoter during transcription elongation. It remains to be investigated whether this model applies to all genes with distal enhancers or only a subset thereof. E: enhancer, P: promoter, red line: dynamic loop extrusion during elongation. 5

6 Supplemental Materials and Methods RNA-FISH We fixed cells in.85% formaldehyde for min at room temperature, and stored them in % ethanol at 4 until further processing. We performed single-molecule RNA FISH on samples as described previously (Levesque and Raj, Raj et al. 8, Raj and Tyagi ). Kit 5 end intron probe conjugated to Alexa594, end probe conjugated to Cy5, and exon probe conjugated to Cy were simultaneously hybridized to samples, followed by DAPI staining and wash steps performed in suspension. Samples were cytospun onto slides for imaged on a Nikon Ti-E inverted fluorescence microscope using a x Plan-Apo objective (numerical aperture of.4), a cooled CCD camera (Pixis 4B from Princeton Instruments), and filter sets SPv (Chroma), SP4v (Chroma), Custom filter (Omega), and v (Chroma), for Cy, Cy5, Alexa594, and DAPI, respectively. We took 45 optical z-sections at intervals of.5 microns, spanning the vertical extent of cells, with s exposure time for Alexa594, Cy, and Cy5, and ms for DAPI. For image analysis, we manually identified cell boundaries and counted RNA spots using custom software written in MATLAB (Raj et al. 8, Raj and Tyagi ). Sequencing track We took snapshot images of sequencing tracks on Fig. SA from public data for DHS, HK4me (Wu et al. ), and RNAPII (Hsiung et al. unpublished) on PSU Genome Brower on Mouse July (NCBI/mm9) Assembly. The CD4 enhancer (-8kb) is defined by capture-c and variety of enhancer marks (Hughes et al. 4). 6

7 Primer List PT-RT-qPCR (β-globin) intron (a) F 5 -CTT CTC TCT CTC CTC TCT CTT TCT CTA ATC- R 5 -AAT GAA CTG AGG GAA AGG AAA GG- intron /exon junction (b) F 5 -GCC TGC AGT ATC TGG TAT TTT TG- R 5 -TGA AAT CCT TGC CCA GGT G- ChIP (β-globin locus) βmaj promoter F 5 -CAG GGA GAA ATA TGC TTG TCA TCA- R 5 -GTG AGC AGA TTG GCC CTT ACC- βmaj exon F 5 -AAC GAT GGC CTG AAT CAC TTG- R 5 -AGC CTG AAG TTC TCA GGA TCC- βmaj intron F 5 -CTT CTC TCT CTC CTC TCT CTT TCT CTA ATC- R 5 -AAT GAA CTG AGG GAA AGG AAA GG- βmaj UTR F 5 -GCC CTG GCT CAC AAG TAC CA- R 5 -TTC ACA GGC AAG AGC AGG AA- εy promoter F 5 -ATG ACC TGG CTC CAC CCA T- R 5 -TCT TTG AAG CCA TTG GTC AGC- βh promoter F 5 -AGG TCC AGG GTG AAG AAT AAA AGG- R 5 -ATC TCA AGT GTG CAA AAG CCA GA- C (β-globin locus) LCR-HS probe HS (anochor) HS εy/ βh βh β maj-fragment β maj-fragment IVR HS 5-56-FAM/TCT GCC TGT CCC TGC CTC GTG A/6-TAMSp- 5 -CAG CGT TTT AGT TGG ATA TAG AGT GAA- 5 -GAA CTT GTC AGG GAA TTA CCT AGT ACA G- 5 -GAT CCC TAT TGT CTA CTT TTG CCA G- 5 -CCC ATG TTA CAC CCC ATT ACA AG- 5 -GGC TGG AAC ATC ACT GGA ATA AAT- 5 -CAG TCG AGG AAT GCA ACT GTG A- 5 -AAG ACT AAA AAT CCC AGA TTG ATT CC- 5 -ACT ACC TAA CTC TCA AAA ATC TGT GTG A- C (ERCC) ERCC probe ERCC_ ERCC_ FAM/TCT AGA GCC AAA CTC TCC AGC CAC CAC TTC/6-TAMSp- 5 -GCA GTG AAA ACA CAA CAC AGT TAA TAT G- 5 -GCA GCC ACC GAC TTG GAT- PT-RT-qPCR (Kit) +4. F 5 -GGC TGG AAA CCA CTG CCT TA- R 5 -AGC CTT GCC TGT GCT TAA AGC- +9. F 5 -CCG GGT GGG CCT CAG T- R 5 -GGC ATG GGC TTA CAG TGT CA- +.9 F 5 -TAT CCC TAC CTG GCC TGG AAC T- R 5 -AGA CTG GCA TAG TGG ATC ACG G- +. F 5 -GAC ACA GGC TTG CTG TCT TC- R 5 -AGC CAT CTC TAC TCG CCT TC-

8 +6.9 F 5 -TGC ATG CTC CGT ACA CCA A- R 5 -CCT CCA ATT TGC AGC CTG TAT T- +4. F 5 -AGG CTG GCA TCA CCA TCA AA- R 5 -GCC CAC AGG ATG GAA GAA AGA- +4. F 5 -GCT CTG GAA CAT CAT GGT AGG A- R 5 -GCC TAT CTT GCG TCT GGC TAT T- +5. F 5 -TCC CTT CCA AAG GAA ATG TTA ATT A- R 5 -GAA TTG AGG GCC AAT GAG ATA CC F 5 -CCA GGC TTT TGT GCA GTG ATG- R 5 -GCT TCT GGT CCT CTG TGG ATG A F 5 -CTC CCC GCT TCG TTT GAA A- R 5 -TGT GTC CCA CAA ACC AAC CAT- +.8 F 5 -AAC TGA AGC GAG TAC AGC ATT CC- R 5 -TGC TTT TGC TTG TGT ACT GTT AAC TG- ChIP (Kit locus) - F 5 -TGA GTC AAA ATT GGG ACC TGC- R 5 -TCC ATG CAT CAT TGA AAA CCT CT- -46 F 5 -AGA AGG TGC CCC GAG TGT ATA A- R 5 -GCA CAG CTC CTT ACC TTG CAA T- -4 F 5 -GCA CAC AGG ACC TGA CTC CA- R 5 -GTT CTG AGA TGC GGT TGC TG- + F 5 -CAC CTC CAC CAT AAG CCG AAT- R 5 -CTC CTA GAC AAT AAA GGA CAA CCA- +4. F 5 -GGC TGG AAA CCA CTG CCT TA- R 5 -AGC CTT GCC TGT GCT TAA AGC F 5 -AGT GTG TCC CGG TCG TAT GAG A- R 5 -TCT GTG TCA GTG TCT ACG CCC A- +9. F 5 -CCG GGT GGG CCT CAG T- R 5 -GGC ATG GGC TTA CAG TGT CA- +.9 F 5 -TAT CCC TAC CTG GCC TGG AAC T- R 5 -AGA CTG GCA TAG TGG ATC ACG G F 5 -TGC ATG CTC CGT ACA CCA A- R 5 -CCT CCA ATT TGC AGC CTG TAT T- +. F 5 -TGG CAG TCC TGG TTG TAG CA- R 5 -GCT GCA AGC ATG CGA TCA- +4. F 5 -AGG CTG GCA TCA CCA TCA AA- R 5 -GCC CAC AGG ATG GAA GAA AGA- +4. F 5 -GCT CTG GAA CAT CAT GGT AGG A- R 5 -GCC TAT CTT GCG TCT GGC TAT T F 5 -TGG TGT GGA TCC CAA GGT CTA- R 5 -CAG GCT ACC TTC GTG ACC ACT T- +5. F 5 -TCC CTT CCA AAG GAA ATG TTA ATT A- R 5 -GAA TTG AGG GCC AAT GAG ATA CC F 5 -GGA GGA GTT AGG GAA TAT GTC GAT AG- R 5 -GCA GTT CTC CAG GTT GAG TCA GA F 5 -CCA GGC TTT TGT GCA GTG ATG- R 5 -GCT TCT GGT CCT CTG TGG ATG A- +.8 F 5 -AAC TGA AGC GAG TAC AGC ATT CC- 8

9 R 5 -TGC TTT TGC TTG TGT ACT GTT AAC TG- +5 F 5 -CTC CCC GCT TCG TTT GAA A- R 5 -TGT GTC CCA CAA ACC AAC CAT- C (Kit) -4 (anchor ) 5 -GGT TTT GGC TTT GGG AGA CA- -4 end (anchor ) 5 -AAG TGA CCC TGA GCG ATT CC (fragment ) 5 -TGT ACT GTT TGA TTC AGC TTT TCC A (fragment ) 5 -CCA TAG CCA AGG GAA ATA TTC AGA (fragment 4) 5 -ATA CAT TCA TGG TTC CTG CTT AGT GT- -.6 (fragment 4; anchor) 5 -GCC AGT AAC CCT GTC TCG AAA C- +. (fragment 48) 5 -CTC TCC CAC GTG ATA GCC ATA CT- +. (fragment 5) 5 -GCA CAG GAG GTC TTA CTT TGA GAT AA- +.4 (fragemt 5) 5 -ACCAGAACCAGAAAGCATCACTTT- +. (fragment 5) 5 -ATA CTG GAA GTT CAA GGG TGT GC- +4. (fragment 55) 5 -CGG TTT CCT CCC AAG AAG TTC- +.8 (fragment 5) 5 -AGC AAG CTC AGC CTT TAA GAG AAG (fragment 6) 5 -TGA ATT TCT GAT TCA GTG GAA GAC C (fragment 6) 5 -AAT AGA GTA GAT ACA TAT TAA CAA AAG TGA GGT TG (fragment ) 5 -CTT ATC ACT GAC TAC CTC TCT ATC ATG CC- +.4 (fragment ) 5 -AAC TGG CCA ATT ATT TGA GAA AGT TT- Taqman Probe (Kit) 5-56-FAM/AGA TCT CTG /ZEN/CGA GCT CAA GGTTAG CCT GG/IABkFQ FAM/AGA TCT CTG CGA GCT CAA GGT- Digestion efficiency (Kit) Kit 5/5 junction F 5 -TGT GAT AGT TGG AAG AGT TAA GGG AG- R 5 -CCA TTT CCT TAA CCT TCA GCC AT- Kit 5/5 junction F 5 -CTT TCT ACA TTA GAG ATG TCC CCC A- R 5 -CGA GAC AGT ATT GAC CTG CCC T- Kit 54/55 junction F 5 -AAT GAG AAA GTT CCT CCG CTG A- R 5 -AGC CAT TAT GAA CCT GGC CTT- Kit 56/5 junction F 5 -TGG CTT CTT TGA AAC AGA TTT TTT T- R 5 -TCT TTT ATC CCA GTG CTC AGG AG- Kit 58/59 junction F 5 -TGA TGT GCA TAA TGG TGG CAT- R 5 -CCA GTC CAA TCC GAT CTC CA- Kit 6/64 junction F 5 -CCT GGA ACT CCT CCT GTA GAC AA- R 5 -GGA GAC TAG CTG GGT GGT GG- Kit 66/6 junction F 5 -ACC CAG GTC AGA CAG TTC CG- R 5 -AAA AAA CAG ACT GGG ATG AGA GGA- Kit / junction F 5 -TGT GTT TTG CTT GCG GCA- R 5 -ACC TCT ACC TTC ACC CAT GAG G- Undigested control primer (Kit) +4. F 5 -AGG CTG GCA TCA CCA TCA AA- 9

10 R 5 -GCC CAC AGG ATG GAA GAA AGA- PT-RT-qPCR and ChIP (CD4) -.4 F 5 -GAGCCAGAGGGAAGGAGTTC- R CACCATCTTCCCTCCCAAG +9.5 F AGGTAGTGCCCAAGAAGCAA R GGGGAGAAGTGACATCACAAA +9. F AAGGCATGGTTCCTGGTAAA R GCCTCAAAGTTCACAGCACA +9. F CCCTGTCTGTGCCTGTTGTA R GCATCAGGTATGGCCTTCAG +8.5 F ATGAGGGGCTAAGGAGTGGT R CAAGCGATGCCATTTCTGTA +5. F GTGTATTCCGCTGTGGCTCT R AATTTCCTACCCCACCCATC C (CD4) -8 (anchor) 5 -TTC TCT CCT CCT GTC CTC CA AGAACGCCATTCACTTGTCC TGAGCTGAACACAGGAGCTG GCAGAAATCTGTGGCAGTCA TCC CTG TAG GGT AGA GGA GGT AACCTCTGCCCAAACTGAAA- IFT5 promoter 5 -ACACACACATTTAAAAACATAAGTCAT- IFT ACACACACATTTAAAAACATAAGTCAT- RNA-FISH probe kit_prime_intron_ kit_prime_intron_ kit_prime_intron_ kit_prime_intron_4 kit_prime_intron_5 kit_prime_intron_6 kit_prime_intron_ kit_prime_intron_8 kit_prime_intron_9 kit_prime_intron_ kit_prime_intron_ kit_prime_intron_ kit_prime_intron_ kit_prime_intron_4 kit_prime_intron_5 kit_prime_intron_6 kit_prime_intron_ kit_prime_intron_8 kit_prime_intron_9 kit_prime_intron_ aagaaaaggcttgtgagtcc ttcaaaccctgctagcaaag ccaagaaccaagtgagtgac atgaaaagaaccacagccat tactgtcaaaagcttcggtg ttgtttgtcactcctggttc ttggtccttgtcctttgaag agaaggaagcccagcttata gcttgcattgttttccttcc cagacagtcccatgatcaac acgaaagccatgactttcat ttccttccggctgttttaaa gtcctaagttcaaacagcca caagagaggaaacaatggct gcttaaatggagtctggtcc cagtgttgcgctattaggaa ccctcagaacaagtgctaac tgtcaggaatcaaaagcact gatctgaggatccctagagc agtactagctagcctgtgaa

11 kit_prime_intron_ caatgtttctcttcggctgg kit_prime_intron_ agaatgaggtacaacactgc kit_prime_intron_ actgttgcagaatcctgaag kit_prime_intron_4 gtgacaaaacccagcaaatg kit_5prime_intron_ taacaaaaaggaagccgtgg kit_5prime_intron_ caagaaaaggctctccaact kit_5prime_intron_ ctgagtatggctatcacgtg kit_5prime_intron_4 tgcagattgtctctagccta kit_5prime_intron_5 catctgacgccttattgcag kit_5prime_intron_6 aaaacaggtcacagagatgc kit_5prime_intron_ gagatctggaaaacgcagtc kit_5prime_intron_8 gaacactcatttgtgaccct kit_5prime_intron_9 cgtaaaagatcactggggtc kit_5prime_intron_ gctctaaagcagaacaaggt kit_5prime_intron_ ctgaaaaggaggcctgaaac kit_5prime_intron_ caaaaccaaggaggcagaat kit_5prime_intron_ caagtaccttccgtggaaac kit_5prime_intron_4 atccttcgtgatgcaaatgt kit_5prime_intron_5 gctgtaaacccactgtgaat kit_5prime_intron_6 gaatgactagggacttgcac kit_5prime_intron_ caggatcgtaagccagaatg kit_5prime_intron_8 cacagtaggaagagtgttgc kit_5prime_intron_9 attgtgaagccccaagtatg kit_5prime_intron_ ggagccttgcagacatttac kit_5prime_intron_ ctgagtacagaaatggcagg kit_5prime_intron_ gggcaagtgacttattctcc kit_5prime_intron_ acctctaacaggatggactt kit_5prime_intron_4 agtagtggatcctcatagca kit_exons_ tgacaaagtcgggatcaatg kit_exons_ aataattggacacctgtggg kit_exons_ gagatttcccatcacactcg kit_exons_4 cacgtttttgatggtgatgc kit_exons_5 caacagggatagccttgatg kit_exons_6 gagggttcatctttagccac kit_exons_ tgtgacatttgctgatccaa kit_exons_8 cagagttggacaccagaaag kit_exons_9 tacattctggacctgtacgt kit_exons_ tgagcaccatcacaatgatc kit_exons_ atcataaggaagttgcgtcg kit_exons_ tattcatgtgattgcccagg kit_exons_ tattcttgcggatctcctct kit_exons_4 agaagctcagcaaatcatcc kit_exons_5 gcaagtcttcatgacgtcat kit_exons_6 gcttgaatgttggccttttc kit_exons_ cctggaataggatgcaagtc kit_exons_8 ggagctactttgctattggg kit_exons_9 cttcaagtgaactactggcc kit_exons_ ctcccatcattaaggatggc kit_exons_ agagcagagcatcattgttc kit_exons_ caggtcagttgtacagaagc

12 kit_exons_ kit_exons_4 kit_exons_5 kit_exons_6 kit_exons_ kit_exons_8 kit_exons_9 kit_exons_ kit_exons_ kit_exons_ gattccaatgctactcccag atacaaaggctgactaggga cctccaaagtatgaacgcat agtttaacttgtgctccctg gtgacagaagctgttacgtc aatctgtttccctggattgg gtgaaggcaacatacctgaa tcaagtgcaaacgatcactt accagcttactttggaaagg ccacatgtaacgtgacatgt Supplemental References Hughes JR, Roberts N, McGowan S, Hay D, Giannoulatou E, Lynch M, De Gobbi M, Taylor S, Gibbons R, Higgs DR. 4. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat Genet 46: 5-. Levesque MJ, Raj A.. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods : Raj A, Reed E, Welles SL, Santana MC, Silverman JG. 8. Intimate partner violence perpetration, risky sexual behavior, and STI/HIV diagnosis among heterosexual African American men. Am J Mens Health : Raj A, Tyagi S.. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 4: Wu W, Cheng Y, Keller CA, Ernst J, Kumar SA, Mishra T, Morrissey C, Dorman CM, Chen KB, Drautz D et al.. Dynamics of the epigenetic landscape during erythroid differentiation after GATA restoration. Genome Res :

13 Relative PT level Interaction frequency untreated βmaj pro DRB released exon Fraction of input intron int/ex ɛƴ βh βmaj pro exon intron ' UTR UTR Fraction of input ɛƴ βh βmaj pro exon intron ' UTR Fraction of input Lee_Figure S Interaction frequency ɛƴ βh βmaj pro exon intron ' UTR untreated DRB released A β-globin B C Primary transcript RNAPII-ChIP 5 intron int/ex. untreated DRB released (hr) GATA: D LCR C kb HS HS ey βh βmaj βmin HS 6 untreated DRB released 6 β-globin locus

14 Elongation rate (kb/m) Wave distance (kb) m8s Relative transcript level (%) Lee_Figure S A 5 Kit locus (kb) B Time (min) C Time (min)

15 exon intron 5 intron 5 Kit 5 Kit Fraction of cells with intron spot Lee_Figure S A asynchronous DRB DRB DRB 4 DRB 5 H L (kb) Kit PT kinetics after DRB release B 5 intron intron Time (min)

16 Lee_Figure S4 SPT5-ChIP.. SPT5 IgG H L min min Fraction of input min min min min Kit locus (kb)

17 SPT5-ChIP Fraction of input Fraction of input RNAPII-ChIP Fraction of input Fraction of input C Interaction frequency Lee_Figure S5 A 6 Enhancer/Promoter Control Time (min) B Enhancer Promoter proximal. RNAPII IgG. RNAPII IgG Time (min) Time (min). SPT5 IgG. SPT5 IgG Time (min) Time (min)

18 C (enhancer anchor) C PT RNAPII SPT5 RNAPII enhancer anchor promoter anchor Time (min) Time (min) % FA % FA 5 C RNAPII PT Time (min) Lee_Figure S6 A kb Kit locus B kb CD4 locus C kb H L

19 CT % of digestion Kb Marker Kit BAC DNA Digested BAC by Bgl II Ligated BAC DNA Lee_Figure S A B M M 5. bp.. M M.5 (kb) bp C D R² Digestion Efficiency E-4 BAC standard of Kit Fragment Junction (BglII)

20 Interaction frequency Lee_Figure S8 E Anchor: -4 H L P Kit gene min min min min min min Kit locus (kb)

21 Interaction frequency Lee_Figure S9 DRB block & release Crosslinking with % formaldehyde for min Anchor: -4 PolII ChIP C E P Kit locus (kb) Kit gene PolII(N) C min min 4 min min 8 min 5 min

22 Relative interaction frequency Lee_Figure S A kb IFT5 CD4-8 BglII fragment DHS HK4me RNAPII (N) B Promoter Promoter +. min min 4 min min CD4 IFT5

23 Lee_Figure S Model A Model B E P E P Dynamic loop extrusion mrna coding region mrna coding region