We report intron chromosomal expression FISH (iceFISH), a multiplex imaging method for measuring gene expression and chromosome structure simultaneously on single chromosomes. We find substantial differences in transcriptional frequency between genes on a translocated chromosome and the same genes in their normal chromosomal context in the same cell. Correlations between genes on a single chromosome pointed toward a cis chromosome-level transcriptional interaction spanning 14.3 megabases.

1. Single-chromosome transcriptional
profiling reveals chromosomal
gene expression regulation
Marshall J Levesque, Arjun Raj
Systems Biology Lab
UPenn Bioengineering
Nature Methods (2013) doi:10.1038/nmeth.2372
Thursday, April 11, 13

2. Our genome is organized
into pairs of chromosomes
By 718 Bot at en.wikipedia [Public domain], from Wikimedia Commons. http://en.wikipedia.org/wiki/Karyotype
Thursday, April 11, 13

3. Chromosome rearrangements
are a hallmark of cancer
Macville et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res (1999) vol. 59 (1) pp. 141-50
Thursday, April 11, 13

4. Does chromosome structure
affect gene expression?
A HeLa Karyotype
t(13;19)
t(13;19)
Chr 13
Chr 13
q-arm
q-arm
t(6;19)
t(6;19)
Chr 19
Chr 19
Chr 19
Chr 19 Chr 6
Chr 6
p-arm
p-arm q-arm
q-arm
Chr 19
Chr 19
q-arm
q-arm
Chr 6
Chr 6
RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm
p-arm
C
Thursday, April 11, 13

5. Does chromosome structure
affect gene expression?
A HeLa Karyotype
t(13;19)
t(13;19)
Chr 13
Chr 13
q-arm
q-arm
t(6;19)
t(6;19)
Chr 19
Chr 19
Chr 19
Chr 19 Chr 6
Chr 6
p-arm
p-arm q-arm
q-arm
Chr 19
Chr 19
q-arm
q-arm
Chr 6
Chr 6
RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm
p-arm
C
Thursday, April 11, 13

6. Does chromosome structure
affect gene expression?
A HeLa Karyotype
t(13;19)
t(13;19)
Chr 13
Chr 13
q-arm
q-arm
t(6;19)
t(6;19)
Chr 19
Chr 19
Chr 19
Chr 19 Chr 6
Chr 6
p-arm
p-arm q-arm
q-arm
Chr 19
Chr 19
q-arm
q-arm
Chr 6
Chr 6
RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm
p-arm
C
Thursday, April 11, 13

7. RNA counting cannot tell us
where the RNA comes from
A HeLa Karyotype
t(13;19)
t(13;19)
Chr 13
Chr 13
q-arm
q-arm
t(6;19)
t(6;19)
Chr 19
Chr 19
Chr 19
Chr 19 Chr 6
Chr 6
p-arm
p-arm q-arm
q-arm
Chr 19
Chr 19
q-arm
q-arm
Chr 6
Chr 6
RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm
p-arm
C
Thursday, April 11, 13

8. iceFISH assay detects active
genes on single chromosomes
mRNA
Export and
Translation
Spliced mRNA
Exon Exon
Exon Intron
Intron
Introns degraded before
leaving site of transcription
Raj et al. Imaging individual mRNA molecules using
multiple singly labeled probes. Nat Methods (2008)
Thursday, April 11, 13

9. iceFISH assay detects active
genes on single chromosomes
intron
Export and
Translation
Spliced mRNA
Exon Exon
Exon Intron
Intron
Introns degraded before
leaving site of transcription
Thursday, April 11, 13

10. Pseudocoloring provides 20+ unique
tags to trace a chromosome
Intron
A B Chromosome 19
Intron
A B Chromosome 19
5 base colors
5 base colors
2 colors 3 colors
2 colors 3 colors 20 pseudocolors
20 pseudocolors DAPI ATTO488 Cy3
C D E
C DAPI D ATTO488 E
F Alexa 594 G ATTO647N H ATTO700
F
Thursday, April 11, 13
Alexa 594 G I Chr 19
J
ATTO647N H ATT

11. Pseudocoloring provides 20+ unique F G H
Alexa 594 ATTO647N
tags to trace a chromosome
Intron
A B Chromosome 19
I Chr 19
Intron J
A B Chromosome 19
Chr 19
5 base colors
5 base colors
2 colors 3 colors
2 colors 3 colors 20 pseudocolors
4
z (µm)
20 pseudocolors DAPI ATTO488 Cy3
C D 2 E
C DAPI D ATTO488 0
E
16
12
8
0 Mb Chromosome 19 59.13 Mb
y (µm) 4
F Alexa 594 G ATTO647N H ATTO700
PTBP1
EEF2
SLC1A5
PPP2R1A
RPS9
ZNF444
DNMT1
ILF3
UBA2
SUPT5H
FBL
EGLN2
RPS19
TOMM40
MARK4
DHPS
RPL18A
UBA52
ZNF91
EIF3K
F
Thursday, April 11, 13
Alexa 594 G I Chr 19
J
ATTO647N H ATT

12. iceFISH assigns gene activity to
normal or derivative copies of chr19
A HeLa Karyotype B
t(13;19) t(6;19)
Chr 19
Chr 13
q-arm
t(6;19)
Fusion
Chr 19 chromosomes
Chr 19 t(13;19)
Chr 6
p-arm
q-arm
Chr 19
Chr 19
q-arm
Chr 6
0 Mb Chromosome 19 59.13 Mb
p-arm
PTBP1
EEF2
SUPT5H
FBL
EGLN2
RPS19
TOMM40
MARK4
SLC1A5
PPP2R1A
RPS9
ZNF444
DNMT1
ILF3
DHPS
RPL18A
UBA52
ZNF91
UBA2
EIF3K
C
Chr 19
Chr 13
1 Translocation Centromere t(13;19)
t(6;19)
t(13;19) t(13;19) ***
some
Thursday, April 11, 13 0.8 N=50 N=30 ***

13. p-arm
q-arm
Chr 19
Translocations can lead to whole
chromosome increases in expression Chr 19
q-arm
Chr 6
p-arm 0 Mb Chromosome 19 59.13 Mb
PTBP1
EEF2
RPS19
TOMM40
MARK4
DNMT1
ILF3
UBA2
SUPT5H
FBL
EGLN2
SLC1A5
PPP2R1A
RPS9
ZNF444
DHPS
RPL18A
UBA52
ZNF91
EIF3K
C
Chr 19
A HeLa Karyotype B Chr 13
1 Translocation Centromere t(13;19)
t(13;19) t(6;19)
t(6;19)
t(13;19) t(13;19) Chr 19 ***
iceFISH spots per chromosome
Chr 13
q-arm
t(6;19)
0.8 N=50 N=30 ***
*** ***
Fusion
Chr 19 0.6 chromosomes
Chr 19
Chr 6
** t(13;19)
*** ** *** ***
p-arm
q-arm 0.4
* Chr 19 **
0.2
Chr 19 0
MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A RPS9 ZNF444
q-arm
Chr 6 Chr. 13 Chromosome 19 gene name
0 Mb Chromosome 19 59.13 Mb
p-arm gene name
PTBP1
EEF2
SUPT5H
FBL
EGLN2
RPS19
TOMM40
MARK4
DNMT1
ILF3
DHPS
RPL18A
UBA52
ZNF91
UBA2
SLC1A5
PPP2R1A
RPS9
ZNF444
EIF3K
C 1
N=30
t(6;19)
Chr 19
0.8 Chr 13
Translocation Centromere t(13;19)
t(6;19)
t(13;19) t(13;19)
0.6 ***
N=50
Thursday, April 11, 13
N=30 ***

14. q-arm
iceFISH spots per chromo
Chr 19
***
Translocations do not necessarily***lead
**
Chr 19
*** **
0.6
to changes in transcriptional activity
q-arm 0.4
* Chr 6
Chr 19 p-arm 0 Mb Chromosome 19 59.13 Mb
q-arm
Chr 6 0.2
PTBP1
EEF2
DNMT1 DNMT1
ILF3
DHPS
RPL18A RPL18A
UBA52
ZNF91
SUPT5H SUPT5H
FBL
EGLN2 EGLN2
RPS19 RPS19
TOMM40 TOMM40
MARK4 MARK4
UBA2
SLC1A5 SLC1A5
PPP2R1A
RPS9
ZNF444 ZNF444
EIF3K
0 Mb Chromosome 19 59.13 Mb
C p-arm
PTBP1
EEF2
FBL
ILF3
DHPS
UBA52
ZNF91
UBA2
PPP2R1A
RPS9
EIF3K
0
C MTZ1 DIAPH3 RPL18A
Chr. 13
UBA52 ZNF91
Chr 19 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM
UBA2
Chromosome 19 gene name
Chr 13
gene name
1 Translocation
A Centromere
HeLa Karyotype B Chr 19
t(13;19)
Chr 13
t(6;19)
t(13;19) t(13;19) ***
iceFISH spots spots per chromosome
t(13;19) 1 t(6;19)
Translocation Centromere t(13;19)
1
0.8 N=50 N=30 N=30 Chr 19
t(6;19)
***
t(13;19) t(13;19) Chr 13
t(6;19) *** ***
t(6;19)
iceFISH spots per chromosome
***
iceFISH per chromosome
q-arm
0.8 N=50 N=30 0.8 ***
0.6
*** Fusion ***
0.6
** ***
Chr 19
** ***
0.6
chromosomes ***
Chr 19
**
** *** ***
Chr 6 t(13;19)
0.4
** * p-arm
*** q-arm
Chr 19
0.4
*
0.4 **
0.2
0.2 0.2
0 Chr 19
MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A RPS9 ZNF444
Chr. 13
q-arm
Chromosome 19 gene name
to Chr 6 centromere
Chr 6
0 0 Mb 0 Chromosome 19 59.13 Mb
gene name
MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2
p-arm
EIF3K SUPT5H FBL EGLN2 PTBP1
RPS19 TOMM40DNMT1
EEF2 ILF3 DHPS
MARK4 SLC1A5 PPP2R1A RPS9 ZNF444
Chr. 13 Chromosome 19 gene name
Chromosome 19 gene name
PTBP1
EEF2
SUPT5H
FBL
EGLN2
RPS19
TOMM40
MARK4
DNMT1
ILF3
DHPS
RPL18A
UBA52
ZNF91
UBA2
SLC1A5
PPP2R1A
RPS9
ZNF444
EIF3K
1
gene name C
N=30
t(6;19)
Chr 19
1
0.8 N=30 Chr 13
1 t(6;19)
Translocation Centromere t(13;19)
t(6;19)
0.8 t(13;19) t(13;19) ***
osome
Thursday, April 11, 13
0.6 0.8 N=50 N=30 ***

15. Are chromosomes built to
regulate their genes?
EEF2 intron EEF2 mRNA Chr. 19
Thursday, April 11, 13

16. Single chromosome transcription
profiles unravel gene interactions
Correlated Expression Intra-chromosomal (cis) interactions
Nucleus
Gene A
Chr. 19
Gene B
Anti-Correlated Expression
PPP2R1A
TOMM40
Intra-chromosomal (cis) interactions Inter-chromosomal (trans) interactions
SUPT5H
RPL18A
SLC1A5
ZNF444
MARK4
DNMT1
EGLN2
PTBP1
UBA52
RPS19
ZNF91
EIF3K
DHPS
UBA2
RPS9
EEF2
ILF3
FBL
Nucleus Nucleus PTBP1
Uncorrelated Expression
Gene A Gene A EEF2
Chr. 19 Chr. 19 DNMT1
reskin fibroblasts
ILF3
Gene B Gene B DHPS
RPL18A
UBA52
ZNF91
UBA2
EIF3K
SUPT5H
1A
1A
40
40
Thursday, April 11, 13
H
H
A
5
A
5
4
4
4
4
1
1