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.
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 ***