ngs-asia rna-seq based expression analysis of the cho cell protein secretion...
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RNA-seq based expression analysis of the CHO cell protein secretion pathway
Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup; Kristensen, Claus;Andersen, Mikael Rørdam
Publication date:2013
Document VersionPeer reviewed version
Link back to DTU Orbit
Citation (APA):Lund, A. M., Kaas, C. S., Kildegaard, H. F., Kristensen, C., & Andersen, M. R. (2013). RNA-seq basedexpression analysis of the CHO cell protein secretion pathway. Poster session presented at 3rd Annual NextGeneration Sequencing Asia Congress, Singapore, Singapore.
Strategy of in silico resonstruction of CHO cell pathways
Objectives Design and methods for RNA-Seq samples
Perspectives
Reconstructed UPR pathway and RNA-Seq data analysisPP2A/Ppp2r5a
ERdj3QSOX1/Qsox1
JNK/Mapk8PP2A/Ppp2r2a
JEM1/Blzf1OSTcomplex/Stt3a
UGT1/Uggt1Traf2Bax
PERKHrd1/Syvn1
NRF2CNX/CANX
QSOX2/Qsox2Gcn2/Eif2ak4
IRE1α/Ern1Crebrf
Traf6Gsk3b
Wfs1Edem3
HSP72/Hspa2Ikbke
OTU1/Yod1TORC2/CRTC2
Ube4bbeta−actin
PP2A/Ppp2r4Atf6b
GlcIIα/GanabPP2A/Ppp2r1bMKK7/Map2k7
NRF1PTP−1B/Ptpn1PP2A/Ppp2r2c
Casp9Creb3
BtkOsp94/Hspa4l
Derl3IkbkbS2P
Nf−yaAIP/Dab2ip
Calpain1/Capn1PP2A/Ppp2r1a
UBXD8/Faf2Bcl2
Hsp70A1/Hspa1aCyclinD1/Ccnd1
Bak1ERdj2/Dnajc11UBC6/Ube2j2
PrkrirIKKA/Chuk
PP2A/Ppp2r5eTBP
PP2A/Ppp2r2dTcp1Atf3
PPI/FKBP65/Fkbp10OASIS
GlcI/MogsTRIB3Fbxo6
Hsp110/Hsph1Hsp60/Hspd1
Nf−ycGapdh
Climpp63/Ckap4Irs1
NFKB1HSP47/Serpinh1
Casp3PPI/FKBP23/Fkbp7
Myd88YY1
PP2A/Ppp2r5cPP2A/Ppp2r3d
Foxo1Cxcl12
JAMP1/JkampERdj1/Dnajc1
ERp19/Txndc12Ost4/Ost4
HERP2/Herpud2Bbf2h7
OSTcomplex/Dad1Fmo1
UGT2/Uggt2ASK1/Map3k5
AmfrUBX2/Ubxn4eIF2α/Eif2s1
RACK1/Gnb2l1Nf−yb
P5/Cabp2OGT/OgtCalmegin
CREBH/Creb3l3BLIMP1/Prdm1Egasyn/Ces1eTisp40/Creb3l4
NOX2/CybbPP1C/Ppp1r1c
Hsp90aa1Serp1
PP2A/Ppp2cbPP2A/Ppp2caGlcIIβ/Prkcsh
Atf4CHOP/Ddit3
Calpain2/Capn2GADD34/Ppp1r15a
PdiltPP2A/Ppp2r2b
TirapPpp5cEro1lb
HERP1/Herpud1CRT/CALR
GRP94/Hsp90b1p58^IPK/Erdj6
Ppib/CyclophilinBPDI/ERp57/Pdia3
EDEM1Sdf2l1
PDI/Erp44BiP/Hspa5
GRP170/Hyou1PDI/ERp72/Pdia4
EDEM2PPI/Fkbp2/FKBP13
S1PCaspase12/Casp12
Tapasin/TapbpBdnfDerl2
ERO1−L../Ero1lMalectin/Mlec
OSTcomplex/Stt3bPDI/Erp29
PDI/ERp59/P4hbXbp1
PRKR/Eif2ak2Bag1
VAMP−B/VapbJNK/Mapk9
SEC63/Sec63OST48/Ddost
PDI/Pdia5JNK/Mapk10
VimpOSTcomplex/Tusc3OSTcomplex/Rpn1
BI−1/Tmbim6NEF/BAP/Sil1
0.0 0.2 0.4 0.6 0.8 1.0Height
Chaperone/Foldease Apoptosis related None or very low expression Refrence gene
References1. B.G. Hansen et al., Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case, Appl Environ Microbiol. (2011) 77(9):3044-51. 2. H.H. Nour-Eldin et al., USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories, Methods Mol. Biol. (2010) 643:185-200.3. S.S. Tate et al., Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte, FASEB J. (1990) 4(2): 227-231.
RNA-seq based expression analysis of the CHO cell protein secretion pathway
Anne Mathilde Lund1, *, Christian Schrøder Kaas2, Helene Faustrup Kildegaard3, Claus Kristensen2, Mikael Rørdam Andersen1
(1) Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark; (2) Current address: Novo Nordisk, Maaloev, Denmark; (3) Current address: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hoersholm, Denmark(*) [email protected]
List of mouse UniProt ID’s CHO-K1 genome (RefSeq)
BLASTp (aa <-> aa)
List of protein id accession # (GI)
bad/no hits + isoproteins
tBLASTn Mouse ID <-> DNA (±3 RF)
(aa <-> DNA)
CHO-K1 genome in contigs
Proteome*
(GI <-> GI#)
A list of predicted proteins of the secretion
pathway in CHO cells
RNA-seq transcriptome
Literature study of the protein secretion pathway
The caperones CNX, GluII-a and UGT falls in clusters di�eren from ohter chaperones. This could indicate the close cornection between protein fold-ing, ER stress response og apoptosis.
Many of the chaperones commonly related to protein foldin seems to be higly expressed in all cell lines, but a variaion between pro-tein produces vs non-produces, exponential growth vs stationary phase and stressed cells. E.g. the gene encoding CHOP protein is increased in expressed under stress and in stationay phase, which also have shown positive protein production by knock-down.
CHO−K
1_none_e
xp
CHO−K
1_IgG_e
xp
CHO−K
1_IgG_0%_e
xp
CHO−K
1_none_sta
CHO−K
1_IgG_sta
CHO−K
1_IgG_0%_sta
CHO−K
1_IgG_N
aBu_1
CHO−K
1_IgG_N
aBu_2
CHO−D
G44_FVIII−high_F435−1
CHO−D
G44_FVIII−high_F435−3
CHO−D
G44_FVIII−high_1C9−1
CHO−D
G44_FVIII−high_1C9−2
CHO−D
G44_FVIII−high_1C9−3
CHO−D
G44_FVIII−med_K
19
CHO−D
G44_FVIII−med_K
16
CHO−D
G44_FVIII−med_K
61−1
CHO−D
G44_FVIII−med_K
61−2
CHO−D
G44_FVIII−med_K
61−3
CHO−D
G44_FVIII−med_K
28_1
CHO−D
G44_FVIII−med_K
28_2
CHO−D
G44_FVIII−med_K
36_1
CHO−D
G44_FVIII−med_K
36_2
CHO−D
G44_FVIII−med_K
36_3
CHO−D
G44_FVIII−med_K
46
CHO−D
G44_FVIII−none_K
52
CHO−D
G44_FVIII−none_K
50
PP2A/Ppp2r5aQSOX1/Qsox1
ERdj3JNK/Mapk8
PP2A/Ppp2r2aBax
PERKHrd1/Syvn1
Traf2UGT1/Uggt1JEM1/Blzf1
OSTcomplex/Stt3aCrebrf
IRE1../Ern1Gcn2/Eif2ak4
Traf6Gsk3b
OTU1/Yod1IkbkeWfs1
HSP72/Hspa2Edem3NRF2
QSOX2/Qsox2CNX/CANX
PrkrirUBC6/Ncube2/Ube2j2
IKKA/ChukPP2A/Ppp2r5e
TBPPP2A/Ppp2r2d
Tcp1Hsp70A1/Hspa1aCyclinD1/Ccnd1
Bak1ERdj2/Dnajc11
Bcl2UBXD8/Faf2
PP2A/Ppp2r1aS2PIkbkbNf−ya
Calpain1/Capn1AIP/Dab2ip
BtkOsp94/Hspa4l
Derl3PP2A/Ppp2r2c
Casp9Creb3
PTP−1B/Ptpn1TORC2/CRTC2
Ube4bPP2A/Ppp2r1bMKK7/Map2k7
NRF1Atf6b
GlcII../GanabPP2A/Ppp2r4
beta−actinGlcI/Mogs
Fbxo6TRIB3Atf3
OASISPPI/FKBP65/Fkbp10PPI/FKBP23/Fkbp7
Casp3Myd88
YY1PP2A/Ppp2r5c
Irs1NFKB1
HSP47/Serpinh1Hsp110/Hsph1
Nf−ycHsp60/Hspd1
Climpp63/Ckap4Gapdh
PP2A/Ppp2r3dCxcl12Foxo1
JAMP1/JkampERdj1/Dnajc1
ERp19/Txndc12HERP2/Herpud2
Ost4/Ost4Bbf2h7
OSTcomplex/Dad1Fmo1
ASK1/Map3k5UGT2/Uggt2
AmfrUBX2/Ubxn4
P5/Cabp2OGT/Ogt
eIF2../Eif2s1Nf−yb
RACK1/Gnb2l1BLIMP1/Prdm1Egasyn/Ces1eTisp40/Creb3l4
CalmeginCREBH/Creb3l3
PP1C/Ppp1r1cNOX2/Cybb
GlcII../PrkcshCHOP/Ddit3
Atf4PP2A/Ppp2caPP2A/Ppp2cb
Serp1Hsp90aa1
Calmegin/PdiltPP2A/Ppp2r2b
TirapPpp5c
GADD34/CreP/Ppp1r15aCalpain2/Capn2
S1PCaspase12/Casp12
Tapasin/TapbpBdnfDerl2
ERO1−L../Ero1lOSTcomplex/Stt3b
Malectin/MlecOSTcomplex/Tusc3OSTcomplex/Rpn1
NEF/BAP/Sil1BI−1/Tmbim6JNK/Mapk10
VimpJNK/Mapk9
VAMP−B/VapbSEC63/Sec63
PDI/Pdia5OST48/DdostPRKR/Eif2ak2
Bag1PDI/Erp29
Xbp1PDI/ERp59/P4hb
PPI/Fkbp2/FKBP13EDEM2EDEM1Sdf2l1
PDI/Erp44PDI/ERp72/Pdia4
ORP150/GRP170/Hyou1BiP/Hspa5
Ero1lbHERP1/Herpud1p58^IPK/Erdj6
Ppib/CyclophilinBPDI/ERp57/Pdia3
CRT/CALRGRP94/Hsp90b1
0 4000 Value
0300
Color Key and Histogram
Coun
t
PERK-BiP
BiP
unfolded proteins
PERKdimerize
and +P
PERK-P
eIF2α -P
+P
eIF2α
Translation
Translation
NRF2 Transcription of AARE elements
p58IPK
(Erdj6)p52rIPK
Grp78va
AFT4
- P
GADD34
CHOP(GADD153)
Bcl2
Low levelsNFkB
Trancription ERS elements
BAG-1
BAX/BAKDR5TRB3
ERO1
ATF6
PP1C
BiP
unfolded proteins
ATF6monomerization+ glycosylation
ATF6translocation
to Golgi
WFS1
ATF6SP1SP2 proteolysis
ATF6 p50NF-Y, YY1TBP
XBP1s
Trancription ERS elements
pro-survival factorsBiP, PDI, EDEM, IRE1, XBP1, Bcl2
ATF6 p50
IRE1-BiP
BiP
unfolded proteins
IRE1αdimerize
and +P
IRE1α-P
sXBP1(mRNA)
uXBP1(mRNA)
?ligase
B1-1PP2A
CRT
UPR elements
ERAD factors
ERS elements
JNK
Apoptosis
after prolonged activation
after prolonged activation
RANC
B1-1
- P
+P
RACK1
COPII
ATF3
TRAF2
IRE1α-TRAF2 ASK1
Atk, PP5
Bcl2
BAX/BAK
CAPN1/2
CASP12
JNK
Apoptosis
MKK7
AIP1
ATF3 B1-1HSP72
CHOP(GADD153)
IKKNFκB
+P
+P
+P
+P
+P
The genes there are not ex-pressed or at very low levels across all samples are com-monly related to be tissue speci�c in mouse.
Proteins associated or linked to UPR pathway were identi�ed by manually curate available litterature on mouse models and cells lines. Furthermore, was the know interactions and dynamics of proteins in the UPR pathway mapped. Found proteins were used to iden-tify CHO-K1 genes of the UPR pathway.
Identi�ed genes and mRNA of CHO-K1
Protein known to be involved in the apoptosic pathway and have a direct interaction is also seen to cluster across the samples in the RNA-Seq data. However, apoptosis can be induced trough di�erent path of the UPR pathway dependend on the cause of stress, which is likely the reason for some of theapop-tosis genes to clustered more closely to chaperones and fold-ing proteins
First a study was initiated foridenti�yng mouse proteins inthe litterature associated with the secretionpathway and the ER stress responses. Amino acid sequences were used for BLASTp against the available RefSeq CHO-K1 genome. The identi�ed genes could be used for extracting parts of the mapped RNA-seq data. However, only mapped genes
The CHO-K1 genome was se-quencing recently and the an-notation is at present insu�-cient, so more than 4 mio. reads was form each sample not possible to map.
Control of mapping IGV5
exp. growth
CHO-K1_none CHO-K1_IgG
stationaryexp. growth
control1 samples 1 samples
CHO-DG44_FVIII
none medium high
5 samples 11 samples 2 samples
exponential growth
• RNA was extracted under di�erent growth condidations and treatment from three di�erent CHO cell lines
• Paired-end RNA sequencing was performed by AROS a/s on Illumina HiSeq 2000 platform with a sequencing depth of min. 35 mio reads
NaBu
2 samples
stationary
0%NEAA control 0%NEAA
1 sample 1 sample 1 sample1 sample
Resync paired-end readscmpfastq3
Unique single reads
Commonpaired-end reads
Mapping of readsTophat24
Cluster analysisSpearman correlation8
Di�erential expressionEdgR7
Normalization of counts between samples
EdgR7
Filtering and trimmingPrinseq-lite2
QC of readsFastQC1
Work�ow of RNA-Seq data handling and analysis
Overview of samples
• Reconstruction of the complex cellular machineries of the early protein secretion pathway and unfolded protein responses by employing legacy knowledge of mouse• By using RNA-seq data, a di�erential gene expression analysis of the constructed CHO secretion pathway would provide a unique possibility for identi�cation of active components to increase the productivity of recombinant proteins.
IntroductionThe Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant thera-peutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly un-derstood cellular processes that control and limit recombinant protein production and secretion, a system-wide study was initiated to identify possible engineering targets relevant for therapeutic protein production.
This preliminary study shows the possibilites for using RNA-seq data and cluster analysis to identify new gene clusters based on biological gene expression behaviour that can lead to a greater biological understading of the important industrial cell CHO-K1. n be used for identifying genetic targets for improvement of protein production by overexpression transcription fac-tors og create knock-downs of growth inhibiting.