transcription microvariability detection by ngs institute of immunology centre de recherche...
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Transcription microvariability detection by NGS
Institute of Immunology
Centre de Recherche Public-Santé
National Public Health Laboratory
Luxembourg
Fleur A. D. Leenen, Oliver Hunewald, Sara Vernocchi, Stephanie Schmitz, Regina Brunnhöfer, Anne Molitor, Jonathan D. Turner and Claude P. Muller
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
human GR gene 10 exons• 9 translated exons9 alternative 1st exons• All 5’UTR• Removed after splicing• ATG translation start in exon 2• Translational regulationCpG island• Methylation• Environmental influence on
expression
The NR3C1 gene
Turner et al., Biochem Pharmacol 2010Cao-Lei et al., Hum Genet 2011
A.
B.
Variable expression• 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
Complex 5’ UTR involved in the regulation of the GR transcription
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
Exon 2 –GR total
-actin
1-B / 16
1-D / 14
1-G / 111
1-E / 15
1-C3
1-CTotal
1-C2
1-F / 17
1-B
1-C3
1-C2
1-Ctotal
1-D
1-E
1-F
1-H
GRtotal
b-actin
Turner and Muller, J Mol Endo 2005
Tissue specific expression
5’RACE adapted to NGS
Daudi
A549
T cells
MCF-7
100nM Dex
100U IFNγ
10μM AZA
Cell Line + StimulusMultiple Cell Lines
1. Dephosphorylation
2. Cap removal3. Ligation RNA oligo
Exon 1x Exon 2
Exon 1x Exon 2
Oligo Exon 1x Exon 2
Oligo Exon 1x Exon 2
4. Gene specific RT
5. 1st round PCR
6. Nested PCR
ShearingLibrary preparation
Emulsion PCR
NGS Sequencing(PGM Ion Torrent,life technologies)
Tot. RNA extraction
Region of Interest: 27kbCoverage: 20xTheoretical read number: 540000Practical read number: 250000
Turner et al., Biochem Pharmacol 2010
Data Analysis
NGS data
Read SelectionRemove ligated oligo sequenceMapping & TSS position retrieval
(TopHat + own script)
Selected TSSs Occurrence / Frequency
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
Normalisation (TMM)
Oligo Exon 1x Exon 2
Control Gene: ADRB2R only 1 exon Highly reproducible Identical TSS selection
Technical Confirmation
B.
A. C.
99.82%99.75%99.83%
Multiple Cell Lines: Reproducibility – A549
B.A.
17.26% 17.42% 0.64% Seq/TSS 0.54% Seq/TSS
78.08%
17.26%
78.59%
17.42%
Proximal Promoter:1D, 1J, 1E, 1B, 1F and 1C
78.07%
78.59% 0.82% Seq/TSS
0.81% Seq/TSS
TSS’s of A549 Replicate 1
TS
S’s
of A
549
Rep
licat
e 2
Fre
quen
cy
(%)
TSS Variability is Reduced on Stimulation
Daudi: High Variability
TSS’s
62.88%
34.30%
71.81%
27.02%
11.49% 84.99%
11.05%
86.44%
TSS’s of Dex Replicate 1
TS
S’s
of
Dex
Re
plic
ate
2
11.05% 11.49% 0.53% Seq/TSS 0.82% Seq/TSS
84.99
86.44% 1.03% Seq/TSS
1.01% Seq/TSS
B.A.
Daudi + Stimuli• Dexamethasone• 5-aza-2’ deoxycytidine• Interferon-Ɣ
Reproducible Variability Altered TSS’s selection
TSS Variability is Reduced on Stimulation - 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.
LNSI, CRP-Santé, Luxembourg:
Claude P. MullerJonathan D. TurnerSara VernocchiOliver HunewaldSteffi SchmitzRegina BrunnhöferAnne Molitor
Funding:
Acknowledgments
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