Supplemental Materials to

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights to its

regulation

Authors

Martin S. Buckley1*, Hojoong Kwak1*, Warren R. Zipfel2, and John T. Lis1

1 Department of Molecular Biology and Genetics

2 Department of Biomedical Engineering

Cornell University, Ithaca, NY, 14853, USA

* These authors contributed equally to this work.

Contents

Supplementary Equations …… 2

Supplementary Materials and Methods …… 3 - 9

Supplementary Figures …… 10 - 16

Supplementary References …… 17

1

Supplementary Equations

Derivation of the Pol II escape constant kel

The kinetic constant of escaping Pol II from the pause (kel) is derived as follows. If

we assume a short time interval Δt, and let [Pol II]pr the amount of Pol II at the pause,

then the amount of Pol II from the pause into elongation is

(Escaping Pol II) = kel [Pol II]pr Δt …… (1)

During this Δt time interval, escaped Pol II molecules travel into the gene body and

cover the region from TSS to vΔt, where v is the elongation speed of Pol II. Amount of

Pol II in this region can be derived from the Pol II density λ as,

(Escaped Pol II) = v ⋅λ Δt …… (2)

where λ can be calculated from the gene body Pol II level ([Pol II]gb) and the gene length

(L) as [Pol II]gb / L.

From the comparison of equations (1) and (2), the kinetic constant of pause escape is,

ke = v [Pol II]gb / L [Pol II]pr …… (3)

Derivation of the Pol II termination constant kt

The kinetic constant of terminating Pol II (kt) is derived from the steady state

assumption of free short nascent RNA. The production of the free short RNA is from

terminating Pol II and its associated nascent RNA on chromatin. The production rate is

then

kt [RNA]ch …… (4)

where [RNA]ch is the amount of short nascent RNA on chromatin. If we assume a first

order decay of the free short nascent RNA with the decay constant kd, the decay rate is

kd [RNA]fr …… (5)

where [RNA]fr is the amount of free short nascent RNA.

Under the steady state assumption, (4) = (5) and the kinetic constant of termination is

derived as,

kt = kd [RNA]fr / [RNA]ch …… (6)

2

Supplementary Materials and Methods

Generations of the transgenic fly lines

We generated LacO tagged Hsp70 transgene line pCasperII(attb)-LacO-SCS-

Hsp70-SCS’ as described in detail in this section. The fly crosses that expresses

paGFP,eGFP, and RFP tagged Pol II subunits under UAS promoter control are driven by

a salivary gland specific GAL4 driver line 6979 (Bloomington stock center). The eGFP-

Rpb3 and mRFP-Rpb3 lines are from previous studies(Yao et al. 2006; 2007). We also

generated the Rpb9-paGFP and mCherry-LacI line as described below.

For the pulse chase experiments, we used heterozygous w1118; pCasperII(attb)-

LacO-SCS-Hsp70-SCS’/6979; mCherry-LacI/Rpb9-paGFP. For the control experiments

in Supplementary Fig. 1, w1118 pCasperII(attb)-LacO-SCS-Hsp70-SCS’/6979; mCherry-

LacI/eGFP-Rpb3, in Supplementary Fig. 2, w1118 6979/+; Rpb9-paGFP/RFP-Rpb3, and in

Supplementary Fig. 3, w1118; pCasperII(attb)-LacO-SCS-Hsp70-SCS’/6979; mCherry-

LacI/Rpb9-paGFP were used.

Hsp70 transgenic fly line: The Hsp70 gene region (-252 to +2439) was PCR amplified

from the 56H8 plasmid(Moran et al. 1979) using primers JL15_Hsp70-F/R (sequences

listed below) tagged with HindIII and SpeI restriction sites, and cloned into pBSIIKS

vector (Agilent Technologies) creating pBSIIKS-Hsp70. The SCS/SCS’ boundary

elements were amplified from w1118 flies using primer pairs JL50_SCS-F/R and

JL45_SCS’-F/R tagged with ApaI/HindIII sites and SpeI/XbaI sites respectively. The

PCR products were sequentially cloned into pBSIIKS-Hsp70 to generate pBSIIKS-SCS-

Hsp70-SCS’. LacO repeats (256x) were digested from the vector pSV2-dhrf-

8.32(Robinett et al. 1996) and cloned into the pSTBlue-1 (Millipore) using XhoI/SalI

sites creating pSTBlue-1-LacO. The SCS-Hsp70-SCS’ fragment was digested from

PBSIIKS-SCS-Hsp70-SCS’ and cloned into pSTBlue-1-LacO using ApaI/SpeI sites to

create pSTBlue-1-LacO-SCS-Hsp70-SCS’. Internal restrictions sites for SphI/NotI were

introduced to pCasperII(attb) vector (Gift from Dr. Daniel Barbash’s Lab, Cornell

University) by inserting annealed oligos (JL93_Insert-F/R) between BamHI/BglII sites,

3

creating pCasperII(InsertF/InsertR). The LacO-SCS-Hsp70-SCS’ fragment was digested

with pSTBlue-1LacOSCSHsp70SCS’ and cloned into pCasper II(InsertF/InsertR) using

SphI/NotI sites creating pCasperII(InsertF/InsertR)-LacO-SCS-Hsp70-SCS’. The attb

fragment was digested from the vector pCasper II(attb) and cloned into the

pCasperII(InsertF/InsertR)-LacO-SCS-Hsp70-SCS’ using NotI sites to generate

pCasperII(attb)-LacO-SCS-Hsp70-SCS’. This final plasmid was transformed into the

Drosophila genome using the PHiC31 platform line attp16 (Genetic Services Inc.). The

primer sequences are listed below.

JL15_Hsp70-F: 5’-GCCCAAGCTTCGAATATTCTAGAATCC -3’

JL16_Hsp70-R: 5’-GCCCACTAGTCTAATTGTATCGTAAGAC-3’

JL50_SCS-F: 5’-TCCCGGGCCCAGCAATTAAATTGTTGCTTGGC-3’

JL32_SCS-R: 5’-TCCCTAAGCTTGAATATGCTCTTTAAATCCCAG-3’

JL45_SCS’-F: 5’-TCTGTTCTAGAGGCAGATTTGGGTCCGTCCATG-3’

JL26_SCS’-R: 5’-CCTGTACTAGTCTCGACAACTAACAGAACAGAT-3’

JL93_Insert-F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGTGAGCA

AGGGCGAGGAGCTGT-3’

JL94_Insert-R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTGCTTGTAC

AGCTCGTCCATGCCG-3’

Rpb9-paGFP transgenic fly line: The paGFP gene was PCR amplified from the

mPAGFP-pRK5 plasmid(Patterson and Lippincott-Schwartz 2002) using primers

paGFP-F/R tagged with SacI and AgeI sites and cloned into a Gateway (Invitrogen)

based UAST P-element insertion vector, pTWG (Drosophila Genomic Resource Center),

creating pTW-paG. The Rpb9 cDNA was amplified from Drosophila cDNA (Open

Biosystems) using primers Rpb9-F/R compatible with the Gateway system and the Rpb9

coding region was cloned into the pDONR221 vector using the Gateway system

generating pDONOR221-Rpb9. The clone was transferred to pTW-paG generating the

construct pTW-Rpb9-paG. The Rpb9-paGFP fusion construct was introduced into the

Drosophila germ line by P-element mediated transformation (Best Gene Inc.). The

primer sequences are listed below.

4

paGFP-F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGTGAGCAAG

GGCGAGGAGCTGT-3’

paGFP-R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTGCTTGTACAGC

TCGTCCATGCCG-3’

Rpb9-F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGACGACTGCCTT

TGATGC-3’

Rpb9-R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTACTCCGTCCAACGGTG

GG-3’

mCherry-LacI transgenic fly line: The minimal Sgs3 promoter (drives gene expression

in salivary gland) was PCR amplified from w1118 flies using primers Sgs3-F/R tagged

with PstI and XhoI sites and cloned into pCaSpeR-4 vector(Thummel and Pirrotta 1992)

creating pCaSpeR-Sgs3. mCherry was PCR amplified from the pRSET-B-mCherry (Gift

from Dr. Roger Tsien’s lab, UCSD) using primers mCherry-F/R tagged with XhoI and

EcoRI sites, and cloned into pAFS144 vector (contains LacI ORF)(Straight et al. 1998)

creating pAFS-mCherry-LacI. The mCherry-LacI fragment was digested from the pAFS-

mCherry-LacI and cloned into pCaSpeR-Sgs3 using XhoI/XbaI sites creating pCaSpeR-

Sgs3-mCherry-LacI. The mCherry-LacI fusion construct was introduced into the

Drosophila germ line by P-element mediated transformation (Best Gene Inc.). The

primer sequences are listed below.

Sgs3-F: 5’-GAGACTCGAGGTTTTTTACTTAATTCTCCA-3’

Sgs3-R: 5’-GATGCTGCAGTCAAACAAAGGGGAGAAGGCTTGTGT-3’

mCherry-F: 5’-GCATCTCGAGATGGTGAGCAAGGGCGAGGAGGATA-3’

mCherry-R: 5’-GCATGAATTCCTTGTACAGCTCGTCCAT-3’

Fluorescence Decay After Photoactivation (FDAP) of polytene nuclei.

Intact Drosophila salivary glands were dissected from third instar larvae and

transferred to a MaTek glass bottom dish (P35G-1.0-14-C) containing Grace’s media

(Sigma-Aldrich) and a glass coverslip was placed on the sample to reduce movement of

the glands. For drug experiments, glands were transferred to 500 nM Flavopiridol

5

(Sigma-Aldrich) diluted in media. Laser scanning confocal microscopy of salivary

glands was carried out on a Zeiss 710 microscope using a Zeiss 63x C-Apochromat

objective (numerical aperture 1.2, water immersion). The RFP-LacI tagged Hsp70

transgene was identified using a 561 nm laser. Samples were photoactivated using a

circular region of interest (ROI) limited to the dimensions of the RFP-LacI spot using a

405 nm laser. The fluorescent of both the RFP-LacI and paGFP-Rpb9 was imaged using

561 and 488 nm lasers every 30 sec for 12 min. To confirm that the Hsp70 gene was

targeted, an objective pre-heated to 37°C (Bioptechs) was used to heat-shock samples for

20 min. The images were analyzed with ImageJ.

Correction of the background genomic Pol II signal.

The FDAP measurement within a ROI can have non-Hsp70 Pol II background

signal arising from nearby genes. To correct for this background, we used a mixed linear

decay model for Pol II signals from adjacent genes (Supplementary Fig. 6). We note that

there is little other Pol II in the immediate vicinity (+/-30 kb) of the transgene

site(Markstein et al. 2008); the promoter GRO-seq signals beyond this are also relatively

modest, and gene body GRO-seq signals decay after the completion of the transcription

in each gene. For each gene within 150 kb from the Hsp70 transgene, Pol II level, gene

length, and the distance from Hsp70 transgene were obtained using GRO-seq datasets

(Supplementary Fig. 6a, 6b). Then assuming that Pol II signals clear from the gene

bodies with the elongation rate of 1.5kb/min, we derived a linear decay function for each

gene (Supplementary Fig. 6c). We introduced a resolution parameter, and took the

distance-dependent Gaussian-weighted (exp(-distance2/2/resolution2); Supplementary Fig.

6d) sum of individual decay functions as the background signal decay function

(Supplementary Fig. 6e). We fitted each FDAP time-course curve to a two-component

model composed of mixed linear decay with a resolution parameter, and an exponential

decay with a half-life parameter. For the best fit resolution, the grid search algorithm was

used to find the resolution and half-life pair applying the least square method. The search

space was [5, 100]×[0.05, 50] and the grid size was 0.05×5 (min×kb). First, the

parameter pairs were limited to those that have the sum of squares for error (SSE) within

6

the +5% margin of the least SSE. Then, the pair with least resolution parameter was

selected. Finally, the background component of the mixed linear decay model with the

resolution parameter was subtracted from the FDAP measurement, and was fit to an

exponential decay model. The highest and the lowest values from each condition were

removed to obtain the average half-lives and the kinetic constants.

Biochemical analysis of steady state kinetics.

The rate constant of elongating Pol II from pausing (kel) was derived from GRO-

seq and PRO-seq data in Drosophila S2 cells at the Hsp70 gene:

kel = vλ / [Pol II]pr

where v = Pol II elongation speed (kb/min), λ = gene body Pol II density (reads/kb), [Pol

II]pr = level of promoter proximal Pol II (reads). The kinetic constant of Pol II termination

(kt) was determined from nascent RNA fractionation in S2 cells:

kt = kd ([RNA]fr / [RNA]ch)

where [RNA]ch = nascent RNA in chromatin fraction, [RNA]fr = nascent RNA in free

fraction, kd = free nascent RNA decay constant (min-1) (see Supplementary Equations for

the derivations of the constants).

Nascent RNA fractionation.

Nascent RNA fractionations were performed as described previously(Wuarin and

Schibler 1994), with minor modifications. Briefly, 0.5~1×107 S2 cell was lysed directly

in the 1 M urea lysis buffer and fractionated by ultracentrifugation for 20 min at 45,000 g.

RNA from supernatant fraction was phenol-chloroform extracted. Chromatin pellet was

resuspended in Trizol (Ambion) and disrupted by short bursts of sonication, followed by

RNA extraction for ligation mediated qRT-PCR (Supplementary Fig. 5). A short in

vitro-transcribed spike-in RNA from an Arabidopsis gene (RCP1) sequence (5×10-2 fmol)

was added before ethanol precipitation as a normalization control. Each RNA fraction

was hybridized to a mixture of biotin labeled DNA probes complementary to Hsp70 and

RCP1 sequences, and specific RNA was enriched as described previously(Rasmussen

7

and Lis 1995). RNA was sequentially treated by 3’ RNA adaptor ligation (T4 RNA

ligase I; NEB), 5’ phosphate dependent exonuclease (Terminator; Epicentre), Tobacco

acid pyrophosphatase (TAP; Epicentre), 5’ RNA adaptor ligation, and reverse

transcription (Superscript III; Invitrogen) following the manufacturers’ instructions.

Hsp70 and RCP1 products were quantified by qPCR using primers that span across the

insert and adaptor junctions. The qPCR levels of 16 different Hsp70 products of different

lengths (25-40 nt) were first normalized to RCP1 level. Then we take the average the 16

normalized values as [RNA]fr or [RNA]ch levels. This was repeated up to ~10 biological

replicates and the average and the standard deviations of the biological replicates were

used for further kinetic analysis. RNA adaptor and DNA primer sequences are listed

below.

Hybridization probe for Hsp70 enrichment

Hsp70-probe: 5’-GCTTACGCTTCGCGATGTGTTCACTTTGCTTGTTTGAAT

T/3BioTEG/-3’

RNA adaptor sequences.

5’ RNA adaptor: 5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’

3’ RNA adaptor: 5’-pUCGUAUGCCGUCUUCUGCUUGU/invdT/-3’

5’ adaptor-insert junction primer.

Hsp70-F1: 5’-TCCGACGATC ATTCTATTCAAA-3’

3’ adaptor-insert junction primers.

Hsp70-R25: 5’-CGGCATACGATTCACTTTGC-3’

Hsp70-R26: 5’-GGCATACGAGTTCACTTTGC-3’

Hsp70-R27: 5’-GGCATACGATGTTCACTTTGC-3’

Hsp70-R28: 5’-CGGCATACGAGTGTTCACTTT-3’

Hsp70-R29: 5’-CGGCATACGATGTGTTCACT-3’

Hsp70-R30: 5’-CGGCATACGAATGTGTTCA-3’

Hsp70-R31: 5’-CGGCATACGAGATGTGTTCA-3’

Hsp70-R32: 5’-GGCATACGACGATGTGTTCA-3’

Hsp70-R33: 5’-GCATACGAGCGATGTGTTCA-3’

Hsp70-R34: 5’-GCATACGAAGCGATGTGTTC-3’

8

Hsp70-R35: 5’-GGCATACGATAGCGATGTGTT-3’

Hsp70-R36: 5’-GGCATACGATTAGCGATGTG-3’

Hsp70-R37: 5’-GGCATACGACTTAGCGATGTG-3’

Hsp70-R38: 5’-GGCATACGAGCTTAGCGATGTG-3’

Hsp70-R39: 5’-ACCGGCATACGACGCTTAG-3’

Hsp70-R40: 5’-ACCGGCATACGATCGCTTAG-3’

In vitro transcription template for RCP1 spike-in RNA.

T7template-RCP1-F: 5'-TAATACGACTCACTATAGGGAGATGGTGGACTCT

CCGTTCTTC-3'

T7template-RCP1-R: 5'-CGGATGGATCCACTGATTTGAGGAAGAACGGAG

A-3'

Hybridization probe for RCP1 enrichment

RCP1-probe: 5'-TGATTTGAGGAAGAACGGAGAGTCCACCACCC/3Biotin-3'

5’ RCP1 primers

RCP1-F1: 5'-TGGTGGACTCTCCGTTCTTC-3'

RCP1-F2: 5'-ATCGGGAGATGGTGGACTCT-3'

RCP1-F3: 5'-GACGATCGGGAGATGGTG-3'

3’ RCP1 primers

RCP1-R1: 5'-GGCATACGACGGATGGAT-3'

RCP1-R2: 5'-CGGATGGATCCACTGATTTG-3'

9

Supplementary Figure 1. Identification of the un-induced Hsp70 transgene using LacO tag. a, A complete z-series of polytene nucleus containing the LacO tagged transgenic Hsp70 under uninduced condition, showing mCherry-LacI (top), eGFP-Rpb3 (middle), and merge (bottom). White and pink arrow heads indicate locations of the native (87A/C) and the transgenic Hsp70, respectively. Small panels on right show magnified images of transgenic and native Hsp70 loci. Rpb3 is the Pol II subunit that has been tested and used to track Pol II dynamics reliably in previous studies (Yao et al., 2007, Zobeck et al., 2010). The transgenic Hsp70 locus was identified on site by the mCherry-LacI signal, and the native Hsp70 loci were identified retrospectively after referring to the heat-shock images in panel b. Note the difficulty in locating the endogenous Hsp70 loci without referring to the heat-shock images. b, A complete z-series of the same polytene nucleus containing the LacO tagged transgenic Hsp70 after 20 min heat-shock induction. Scale bars are 10 μm for both panels. c, Quantification of Pol II induction at the transgenic and the endogenous Hsp70 loci (n=3). eGFP-Rpb3 signals after the heat-shock normalized to 87C (left), and fold induction normalized per copy (right). Note that 87A contains 2 copies and 87C contains 4 copies of the Hsp70 gene.

c

0

0.25

0.5

0.75

1

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eG

FP-R

pb3

0

5

10

15

20

25

eGFP

-Rpb

3 fo

ld in

duct

ion

ba

mC

herry

-Lac

IeG

FP-R

pb3

Mer

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Hsp70Transgene

LacI

Rpb

3M

erge

87A/C

20 min heat shock

mC

herry

-Lac

IeG

FP-R

pb3

Mer

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Transgene

LacI

Rpb

3M

erge

87A/C

Uninduced

Hsp70

10

Supplementary Figure 2. Fluorescence labeling of Pol II on native Hsp70 loci by Rpb9-paGFP. a, A complete z-series of polytene nucleus containing the Rpb9-paGFP compared to RFP-Rpb3 under uninduced condition. RFP-Rpb3 (top), Rpb9-paGFP (middle), and merge (bottom). White arrow heads indicate locations of native Hsp70 loci. Small panels on right show magnified images of the native Hsp70 loci. The native Hsp70 loci were identified retrospectively after referring to the heat-shock images in panel b. b, A complete z-series of the same polytene nucleus after 20 min heat-shock induction. Scale bars are 10 μm for both panels.

ba

RFP

-Rpb

3R

pb9-

paG

FPM

erge

Hsp70 at 87A/C

Rpb

3R

pb9

Mer

ge

20 min heat shock

RFP

-Rpb

3R

pb9-

paG

FPM

erge

Hsp70 at 87A/C

Rpb

3R

pb9

Mer

ge

Uninduced

11

Supplementary Figure 3. Labeling of the Rpb9-paGFP by photoactivation on the LacO tagged Hsp70 transgene. a, A complete z-series of polytene nucleus containing the LacO tagged transgenic Hsp70 under un-induced condition before photoactivation. mCherry-LacI (top), Rpb9-paGFP (middle), and merge (bottom). b, A complete z-series of the same polytene nucleus containing the LacO tagged transgenic Hsp70 under un-induced condition after photoactivation. Note that to further illustrate the Rpb9-Pol II banding pattern, this sample was photoactivated throughout the entire nucleus. White and pink arrow heads indicate locations of native and transgenic Hsp70, respectively. The native Hsp70 loci were identified retrospectively after referring to the heat-shock images. Small panels on right show magnified images of transgenic and native Hsp70 loci. c, A complete z-series of the same polytene nucleus after 20 min heat-shock induction and photoactivation. Scale bars are 10 μm for all 3 panels. d, Quantification of Pol II induction at the transgenic and the endogenous Hsp70 loci (n=3). Rpb9-paGFP signals after the heat-shock normalized to 87C (left), and fold induction normalized per copy (right).

0

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Rpb

9-pa

GFP

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Rpb

9-pa

GFP

fold

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ctio

n d

a b

mC

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-Lac

IR

pb9-

paG

FPM

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Pre-photoactivation (uninduced)

mC

herry

-Lac

IR

pb9-

paG

FPM

erge

Hsp70Transgene

LacI

Rpb

9M

erge

87A/C

Photoactivation (uninduced)

mC

herry

-Lac

IR

pb9-

paG

FPM

erge

Photoactivation (20 min heat-shock)

Hsp70Transgene

LacI

Rpb

9M

erge

87A/C

c

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Supplementary Figure 4. Control for photobleaching. a, Fluorescence decay after photobleaching using different imaging intervals to evaluate the photobleaching by laser excitation per each imaging cycle. To correct for photobleaching we compared decay curves with different frequency of excitation cycles. The difference between the two plots reflect photobleaching caused by additional excitation cycles, and are within the margin of error but may result in up to 20% correction of the measured half-lives. The plots were fit to exp(−k0.5t) and exp(−k2.5t) respectively, and normalized to the y intercept of the fit curves. The photobleaching constant was derived from the difference of the two curves, kph = 1.25×(k0.5 − k2.5). b, Corrected half-life measurements of control and Flavopiridol treated salivary gland nuclei using the photobleaching constant from panel a.

0

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alf-l

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in)

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Flou

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inte

nsity

0 2 4 6 8 10 120

0.20.40.60.8

1Imaging interval

0.5 min2.5 min

13

Supplementary Figure 5. Quantitative measurement of Hsp70 short nascent RNA (nasRNA). Short nascent RNA from either chromatin bound or free fraction is analyzed by ligation mediated qRT-PCR. nasRNA derived from different pausing or termination positions are measured using 3’ adaptor-insert junction primers that scan the 3’ ends of the Hsp70 nasRNA from +25 to +40 relative to the TSS.

Multiple pausing or termination positions

RNA extractionHybridization to Hsp70 probeHsp70 specific enrichment

RNA adaptor ligationsReverse transcription

Multiple 3’ positionsHsp70 nasRNA

5’ adaptor-insert junction primer

1’st round amplification with adaptor primers

3’ primer #1

Multiple 3’ adaptor-insert junction primer combinations

3’ primer #2

3’ primer #3

qPCR #2qPCR #1 qPCR #3

TSS Pol II Pol II Pol II

nasRNA

#1 #2 #3

Multiple 3’ position specific measurements

Position #1 Position #2 Position #3

14

Supplementary Figure 6. Correction for the background Pol II signal in fluorescence decay after photoactivation (FDAP). a, Pol II density (GRO-seq) and organization of active genes are shown in the region of ±150 kb from the Hsp70 transgene insert site (53C4 locus on chromosome 2R). b, For each active gene, Pol II is assumed to be evenly distributed throughout the length of each gene body and the gene body Pol II is clearing out with the elongation speed of 1.5 kb/min. c, For active genes, gene body Pol II decay linearly by elongating and terminating at the 3’ ends of the genes. d, The signals from the individual genes are assumed to contribute as the background signal at the Hsp70 transgene as a Gaussian function of the distance over the resolution. e, The background component of the mixed linear decay model is the sum of linear decay curves for individual genes weighted by the distance dependent weight factors. Exponential decay components for a few possible half-lives are also shown. From the possible combinations of the half-life and resolution parameters, the best fit pair is found using a grid search algorithm. f, The position, length, and Pol II levels of the genes near the Hsp70 transgene insert sites are inferred from the GRO/PRO-seq datasets.

Gene Position (kb)

Length (kb)

Pol IIlevel

Tsf3 -147.3 3.8 0.29

CG7786 -140.8 1 0.02

krimp -136 4.8 1.4

CG15709 -120.7 1.6 0.02

fidipidine -112 3.2 0.61

csul -109.7 2.3 0.55

Khc -106 5.8 1.71

CG30324 -87.6 1.1 0.02

JhI-26 -85.2 3.5 1.51

CG7747 -82.3 2.4 0.39

Atg9 -79.7 4 0.95

CG8060 -57 6.8 1.39

CG4282 -52 3.1 0.55

Vha44 -44.8 13 2.13

Hmgs -36.8 5.7 1.49

CG7997 -33.1 4.6 0.4

Nup62 -30.3 1.8 0.61

l(2)k07824 -28.8 2.1 0.71

CG4398 57.2 3.8 0.6

Sema-2a 149.3 20 1.04

f

Find the best fit combination of half-life and resolution

53C4 (chr2R:12,112,957-12,412,957)

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Linear decay models for single genes

0 1 2

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Khc

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Atg9

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Vha44

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CG4398

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Sema-2a

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Time (min)

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ResolutionBackground component Exponential decay component

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Half life

× exp ( -distance2 / 2 / resolution2 )Distance dependent weight factor

a

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15

Supplementary Figure 7. Half life estimation with the background correction of the fluorescence decay after photoactivation (FDAP). FDAP measurements and fits for a, control and b, flavopiridol treatment conditions.

Control (n=9)

Flavopiridol (n=7)

0 5 100

100200300400500 3.5 min

R2=0.9815

0 5 100

200400600800 3.8 min

R2=0.9926

0 5 100

100200300400500600700 5.4 min

R2=0.9847

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100200300400500 7.15 min

R2=0.9986

0 5 100

100200300400500600 4.1 min

R2=0.9772

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50100150200250300350 7.0 min

R2=0.9889

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100200300400500 4.9 min

R2=0.9946

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100200300400 6.8 min

R2=0.9970

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50100150200250300350 5.7 min

R2=0.9946

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100200300400500 4.8 min

R2=0.9945

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200400600800 10.1 min

R2=0.9953

0 5 100

100200300400500600 12.05 min

R2=0.9746

0 5 100

200400600800

1000 24.95 minR2=0.9966

0 5 100

100200300400 13.95 min

R2=0.9891

0 5 100

50100150200250300350 5.15 min

R2=0.9930

0 5 100

50100150200250300 6.85 min

R2=0.9955

a

b

16

Supplementary References

Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N. 2008. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40: 476–483.

Moran L, Mirault ME, Tissières A, Lis J, Schedl P, Artavanis-Tsakonas S, Gehring WJ. 1979. Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein. Cell 17: 1–8.

Patterson GH, Lippincott-Schwartz J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873–1877.

Rasmussen EB, Lis JT. 1995. Short transcripts of the ternary complex provide insight into RNA polymerase II elongational pausing. J Mol Biol 252: 522–535.

Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS. 1996. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. The Journal of Cell Biology 135: 1685–1700.

Straight AF, Sedat JW, Murray AW. 1998. Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast. The Journal of Cell Biology 143: 687–694.

Thummel CS, Pirrotta V. 1992. Technical notes: new pCasper P-element vectors. Drosophila Information Service 71: 150.

Wuarin J, Schibler U. 1994. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol Cell Biol 14: 7219–7225.

Yao J, Ardehali MB, Fecko CJ, Webb WW, Lis JT. 2007. Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation. Mol Cell 28: 978–990.

Yao J, Munson KM, Webb WW, Lis JT. 2006. Dynamics of heat shock factor association with native gene loci in living cells. Nature 442: 1050–1053.

Zobeck KL, Buckley MS, Zipfel WR, Lis, JT. 2010. Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells. Mol Cell 40: 965–975.

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