Transcript TITLE

Transcription microvariability detection by NGS
Fleur A. D. Leenen, Oliver Hunewald, Sara Vernocchi, Stephanie Schmitz,
Regina Brunnhöfer, Anne Molitor, Jonathan D. Turner and Claude P. Muller
Institute of Immunology
Centre de Recherche Public-Santé
National Public Health Laboratory
Luxembourg
Hypothalamic-pituitary-adrenal axis
•
•
•
HPA-axis
 reactions to stress
 many physiological processes
Glucocorticoids
 Nervous & Immune system
 Learning and memory
 Stress response
 …
Glucocorticoid receptor (GR)
 Negative feedback HPA axis
 Ubiquitous presence
 Expression levels vary
 Complicated Structure
The NR3C1 gene
A.
B.
Turner et al., Biochem Pharmacol 2010
Cao-Lei et al., Hum Genet 2011
 human GR gene
 10 exons
• 9 translated exons
 9 alternative 1st exons
• All 5’UTR
• Removed after splicing
• ATG translation start in exon 2
• Translational regulation
 CpG island
• Methylation
• Environmental influence on
expression
Tissue specific expression
1-B / 16 Variable expression
1-B
1-C33
1-C
1-C22
1-C
1-Ctotal
1-C
Total
1-D / 14
1-D
• Hippocampus expresses all
the exons and splice variants
• 1-B, 1-C and 1-H are the
most widely expressed
• 1-D only in the hippocampus
1-E / 15 Complex 5’ UTR involved in the
1-E
1-F / 17
1-F
regulation of the GR transcription
1-H
1-G / 111
Exon
GRtotal2 –GR total
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b-actin
-actin
Turner and Muller, J Mol Endo 2005
5’RACE adapted to NGS
Multiple Cell Lines
Cell Line + Stimulus
Tot. RNA extraction
Daudi
A549
100nM Dex
100U IFNγ
T cells
1. Dephosphorylation
MCF-7
2. Cap removal
3. Ligation RNA oligo
Exon 1x
Exon 2
Exon 1x
Exon 2
Oligo
Exon 1x
Exon 2
Oligo
Exon 1x
Exon 2
10μM AZA
Turner et al., Biochem
Pharmacol 2010
4. Gene specific RT
5. 1st round PCR
6. Nested PCR
Shearing
Library preparation
Emulsion PCR
NGS Sequencing
(PGM Ion Torrent,
life technologies)
Region of Interest: 27kb
Coverage: 20x
Theoretical read number: 540000
Practical read number: 250000
Data Analysis
NGS data
Oligo
Exon 1x
Exon 2
Read Selection
Remove ligated oligo sequence
Mapping & TSS position retrieval
(TopHat + own script)
Selected TSSs
 Occurrence / Frequency
Normalisation
(TMM)
 Selected TSS’s per Cell Line and Conditions
 Sequence Frequencies per TSS
 Shared TSS’s
 Differentially expressed TSS’s
 Use of existing Software packages
(NOISeq)
 Instead of gene, TSS as entity
 Between 2 Cell Lines or Conditions
 ± 25 most important TSS’s
 for differentiation between Cell
Lines/Conditions
Technical Confirmation
A.
C.
B.
99.82%
99.75%
99.83%
 Control Gene: ADRB2R  only 1 exon
 Highly reproducible
 Identical TSS selection
Multiple Cell Lines: Reproducibility – A549
78.59%
 0.82% Seq/TSS
B.
17.42%
78.08%
78.59%
TSS’s of A549 Replicate 2
A.
17.26%
17.26%
 0.64% Seq/TSS
17.42%
 0.54% Seq/TSS
TSS’s of A549 Replicate 1
78.07%
 0.81% Seq/TSS
Proximal Promoter:
1D, 1J, 1E, 1B, 1F and 1C
Multiple Cell Lines: Shared, but DE TSSs
A.
C.
A549
Tcells
Daudi
MCF7
A549
MCF-7
B.
TSS Variability is Reduced on Stimulation
 Daudi: High Variability
Frequency (%)
34.30%
62.88%
27.02%
71.81%
TSS’s
TSS Variability is Reduced on Stimulation - Dex
A.
86.44%
 1.03% Seq/TSS
11.49%
B.
84.99%
TSS’s of Dex Replicate 2
86.44%
11.05%
11.05%
 0.53% Seq/TSS
11.49%
 0.82% Seq/TSS
84.99
 1.01% Seq/TSS
TSS’s of Dex Replicate 1
 Daudi + Stimuli
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•
•
Dexamethasone
5-aza-2’ deoxycytidine
Interferon-Ɣ
 Reproducible Variability
 Altered TSS’s selection
TSS variability is Reduced on Stimulation
A.
C.
B.
Daudi
AZA10
IFN
Dex
Daudi
C
.
Dex
Conclusion
 Majority Reads
 Majority alternative
1st exons
Proximal Promoter  CpG island  transcription factors can
bind at different positions within such a promoter site. The
selection of TSS’s varies according to external influences.
Acknowledgments
LNSI, CRP-Santé, Luxembourg:
Claude P. Muller
Jonathan D. Turner
Sara Vernocchi
Oliver Hunewald
Steffi Schmitz
Regina Brunnhöfer
Anne Molitor
Funding:
Analysis Technique: Manual - TopHat
A.
B.
 Control Gene: ADRB2R
 2 approaches: Manually
→ Similar Results
TopHat