Transcription microvariability detection by NGS

Institute of Immunology

Centre de Recherche Public-SantéNational 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-E1-F1-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 24. 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

TSS

’s o

f A54

9 R

eplic

ate

2

A549

MC

F-7

A. C.

B.

Multiple Cell Lines: Shared, but DE TSSs

Tcel

l sD

aud i

MC

F 7A

549

Freq

uenc

y (%

)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

TSS

’s o

f Dex

Rep

licat

e 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

A. C.

C.

Dau

di

Dex

B.

Dau

diA

ZA1 0

IFN

Dex

TSS variability is Reduced on Stimulation

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

Control Gene: ADRB2R 2 approaches: Manually TopHat

→ Similar Results

Analysis Technique: Manual - TopHat

B.A.