DNA replication : Temporal profiling and origin prediction

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DNA replication Temporal profiling and origin
prediction
Anindya Dutta Neerja Karnani Christopher
Taylor Ankit Malhotra University of Virginia
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Methodology adopted
Label newly synthesized DNA with BrdU at every
2 hrs of S-phase
Purification of BrdU labeled DNA by CsCl banding
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Data verification two biological and two
technical replicates
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Replication Profile for the Entire ENCODE region
52 of the ENCODE region finishes replication by
first four hours of S-phase
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Examples of replication patterns over individual
ENCODE regions

• Slide 6 - Early
• Slide 7 Late
• Slide 8 - panS

Temporally monophasic (significant signal
enrichment at a specific time interval of S
phase)
Asynchronous firing of replication (significant
signal enrichment at more than one time
interval of S phase)
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Examples of early replicating ENCODE regions
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Examples of late replicating ENCODE regions
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Examples of panS pattern of replication across
ENCODE regions
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Temporal profile of Replication for ENCODE in
HeLa adherent
Temporal profile of Replication for Chromosome
21/22 in HeLa adherent (Jeon et al.PNAS
2005,1026419-24)
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Time of replication confirmed by interphase FISH
Replicated
Unreplicated
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Temporally monophasic pattern of replication
Pan-S pattern of replication
Jeon et al.PNAS 2005,1026419-24
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FISH analyses for an early replicating region on
Chromosome 22
FISH analyses for two regions of chromosome 22
showing panS pattern of replication
Jeon et al.PNAS 2005,1026419-24
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Origin Predictions

• For each probe calculate a TR50 by linearly
  interpolating the point at which cumulative
  signal reaches 50 of total from 0-2, 2-4, 4-6,
  6-8, 8-10 time points
• Throw out non-specific probes where at least half
  of the total replication signal did not occur in
  1 time point.
• Smooth the remaining TR50 data (5 window) to
  generate TR50 profile curves.
• Repeat the above procedure for each replicate set
  of chips.
• Construct confidence intervals where troughs in
  the replicate sets TR50 profile coincide.
• Average the replicate sets TR50 curves to
  generate average TR50 and pinpoint troughs to
  make origin predictions.
• Examine the predictions to verify that they fell
  into the confidence intervals from step 5.

Time (hrs)
Chromosome position
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Sequence features of known metazoan replicators
IR
IR
IR
IR
IR
IR
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Beta Globin Locus - Temporal Profile of
Replication
A
B
C
D
E
F
Biological Rep1(BR1)
Technical replicate of BR1
Biological Rep2 (BR2)
Technical replicate of BR2
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Conf. Start Conf. End Difference Avg. Pred.
A 5,065,180 5,092,935 27,755
5,082,768 B 5,178,000 5,219,000
41,000 5,178,000 -5,219,000
C 5,263,570 5,292,750 29,180 5,271,110 D 5,36
6,290 5,460,645 94,355 5,399,952 E 5,543,905 5
,568,880 24,975 5,558,825 F 5,650,750 5,681,275
30,525 5,667,011
A
B
C
D
E
F
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LCR
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A monophasic early replicating area from Chr
21/ENCODE
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ENm005 - Temporal Profile of Replication
ENm005 Replicates with Possible origins
A
B
E
F
C
D
G
H
K
L
I
J
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Conf. Start Conf. End Difference Avg.
Pred. 32,747,964 (1) A 32,864,590 32,885,
852 21,262 32,880,930 (2) B 32,953,565 32,965
,162 11,597 32,946,436 (3) C
32,997,380 33,017,952 20,572 33,011,238 (4) D
33,090,291 33,114,205 23,914 E
33,289,804 33,314,720 24,916 33,291,808 (5) F
33,549,703 33,564,305 14,602 33,553,742 (6) G
33,688,134 33,697,728 9,594 33,700,005
(7) H 33,756,830 33,788,695 31,865 33,83
9,972 (8) I 33,867,360 33,872,982
5,622 33,870,203 (9) J 34,087,453 34,112,618
25,165 34,103,224 (10) K 34,157,799 34,176,347
18,548 34,168,048 (11) L 34,242,782 34,266,6
29 23,847 34,255,806 (12)
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QUESTION
Any explanation for why some segments replicate
early and others replicate late?
( slide 23-28 Chr. 21 and 22 data)
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Low exon density in late replicating segments
Jeon et al.PNAS 2005,1026419-24
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Chromosomal segments replicated after 3 hrs of
S phase exhibit a acute decline in gene density
Jeon et al.PNAS 2005,1026419-24
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AT rich segments enriched around ori sites that
replicate in late S-phase
Chromosome 21
Chromosome 22
Density of AT islands of stated
Density of AT islands of stated
Jeon et al.PNAS 2005,1026419-24
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Gene expression levels decrease with increasing
replication time
Jeon et al.PNAS 2005,1026419-24
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1Mb bin resolution of replication timing data
across chromosome 21 centromeric regions show
late replication
Jeon et al.PNAS 2005,1026419-24
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1Mb bin resolution of replication timing data
across chromosome 22 centromeric regions show
late replication
Jeon et al.PNAS 2005,1026419-24
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Replication profile vs Gene Density for random
picks under ENCODE region
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Euchromatin is associated with early replication
and sensitivity to DNaseI
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