supplemental materials to kinetics of promoter pol ii on hsp70...
TRANSCRIPT
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
Nor
mal
ized
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
ge
Hsp70Transgene
LacI
Rpb
3M
erge
87A/C
20 min heat shock
mC
herry
-Lac
IeG
FP-R
pb3
Mer
ge
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
0.25
0.5
0.75
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Nor
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Rpb
9-pa
GFP
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15
20
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Rpb
9-pa
GFP
fold
indu
ctio
n d
a b
mC
herry
-Lac
IR
pb9-
paG
FPM
erge
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
12
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
5
10
15
20
Ctrl FP
Cor
rect
ed h
alf-l
ife (m
in)
ba
Time (min)
Flou
resc
ence
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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40
-150 -100 -50 0 50 100 150
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RO
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Activegenes
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Linear decay models for single genes
0 1 2
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Khc
0 1 2
0 5 10
Atg9
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Vha44
0 1 2
0 5 10
CG4398
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Sema-2a
Mixed linear decay model
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Rel
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70
Time (min)
60 kb50 kb40 kb30 kb
ResolutionBackground component Exponential decay component
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Time (min)
10 min8 min6 min4 min
Half life
× exp ( -distance2 / 2 / resolution2 )Distance dependent weight factor
a
b
c
d
e
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
0 5 100
100200300400500 7.15 min
R2=0.9986
0 5 100
100200300400500600 4.1 min
R2=0.9772
0 5 100
50100150200250300350 7.0 min
R2=0.9889
0 5 100
100200300400500 4.9 min
R2=0.9946
0 5 100
100200300400 6.8 min
R2=0.9970
0 5 100
50100150200250300350 5.7 min
R2=0.9946
0 5 100
100200300400500 4.8 min
R2=0.9945
0 5 100
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
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