leonardo a. meza-zepeda seminar august... · leonardo a. meza-zepeda dept. of tumorbiology the...
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Leonardo A. Meza-ZepedaDept. of Tumor Biology
The Norwegian Radium Hospital, OUS
Regulatory landscape of bone biology
The Norwegian Radium Hospital
The Norwegian Radium Hospital
Liposarcoma
Adipogenesis
Osteosarcoma
Osteogenesis
Mesenchymal BiologyMyoblast Premyoblast
MSC
Preadipocyte
Adipocyte
Mature
adipocyte
Preosteoblast
Osteoblast
Mature
osteoblast
Prechondrocyte Chondrocyte
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Osteoblast
Osteogenic Differentiation
Alizarin Red staining
Dif
fC
trl
D7 D14 D21
Expression of osteogenic markers
Evidence of differentiation
iMSC3• Immortalized bone marrow hMSCs• Ectopic hTERT expression
21-28 days
Model SystemHåkelien
The Norwegian Radium Hospital
also primary hMSC cultures and osteoblasts
Gene Expression Changes During
Osteogenic Differentiation
The Norwegian Radium Hospital
Undifferentiated
Dif
fere
nti
ate
d 1462 up-regulated
1695 down-regulated
Osteogenic Genes Mesenchymal Genes
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Epigenetic Regulation
The Norwegian Radium Hospital
Modification RELEVANCE
H3K9ac Promoters / active genes
H3K4m3 Promoters / active genes
H3K36m3 Coding regions / active genes
H3K27m3 Repressed genes
H3K9m3 Repressed genes, heterochromatin
H3K27ac Enhancers / active genes
H3 Nucleosome
ChIP-Seq
Fragmentation of chromatin
IP w/ antibody to chromatin
modificationPurify DNA
The Norwegian Radium Hospital
CrosslinkDNA-proteins
Illumina
Sequencing
Sequencing of IP DNAApprox. 30-35 M reads
36 bp single read
Reads
Peaks
Align sequence
reads to genome
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Epigenetic Landscapes
Håkelien, et alThe Norwegian Radium Hospital
Gene Expression and Histone Modifications
The Norwegian Radium Hospital
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Histone Modifications Changes
The Norwegian Radium Hospital
Chromatin changes and gene expression
The Norwegian Radium Hospital
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Identification of Novel Transcriptional
Regulators of Osteogenesis
The Norwegian Radium Hospital
Genes with increased expression
during differentiation
Chromatin that opens up
during differentiation
Promoters and Enhancers
de novo motif discovery
Undifferentiated
Dif
fere
nti
ate
d
Motif Enriched in Promoter Regions
The Norwegian Radium Hospital
TEAD2/ETF
• TEA domain family member 2PURA
• Purine-rich element binding protein AGTF2I
• General transcription factor IIiGTF2IRD1
• GTF2I repeat domain containing 1ZNF148
• Zinc finger protein 148
ZN
F1
48
GT
F2
I
PU
RA
GT
F2
IRD
1
TE
AD
2
Promoter Regions
Candidate Transcription Factors
Williams-Beuren syndromeDeletion of GTFs, cranial facial defects
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Knockdown Affects Osteogenic Differentiation
The Norwegian Radium Hospital
2 siRNAs/gene
Preliminary experiments
Summary
• Approximately 3000 genes differentially expressed during osteogenic differentiation
• Up-regulated genes associated with cell adhesion, extracellular matrix and bone development (�H3K4me3 and �H3K27me3)
• Down regulated genes associated with cell cycle regulation (�H3K27me3, �H3K27ac and �H3K9ac)
• Candidate novel transcriptional regulators of osteogenesis identified by integration of chromatin and gene expression
changes
The Norwegian Radium Hospital
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• Most common primary malignant tumours of bone
• Children/adolescents and older people
• Long bones (arm and leg)
• High grade tumours, aggressive
• Complex genetic changes
Osteosarcomas
What are microRNAs (miRNA)?
• Non-protein coding small RNAs (18-24 nt)
• Involved in all cellular processes
• Regulates protein coding genes post-transcriptionally
5' ...UUAUGGCACUUCAAUUUUGCACA… mRNA
| | | | | | |
3' AGUCAAAACGUACCUAAACGUGU -5’ miRNA
ORF3’ UTR
mRNA
miRNA
mRNA degradation or inhibition of protein synthesis
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miRNA – a role in cancer?
Modified from Caldas and Brenton, Nature Medicine, 2005
miRNA as oncogene miRNA as tumor suppressor
Normal cell
Tumor cell
Mechanisms for miRNA regulation
Iorio, M. V. et al. J Clin Oncol; 2009
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Aim of the study
Identify epigenetically regulated
miRNAs in osteosarcoma
DNA methylationmiRNA expression
Project Strategy
Identify candidate miRNAs that show an abberant methylation
pattern in osteosarcoma cell lines compared to normal samples
Examine methylation pattern of promoter region
in cell lines
Are miRNAs re-expressed if the methylation in inhibited?
Verify methylation level in osteosarcoma patient and normal
samples
Examine effect of transfection with miRNAs in cell lines
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Candidate miRNAs:
• show big difference in methylation level between OS cell lines and normal samples• show high inverse correlation between miRNA expression and methylation level
Methylated miRNAs in osteosarcoma cell lines
osteosarcomanormal
mir-34bmir-34c
BTG4
Økt uttrykk av miRNA etter 5-aza behandling
• Ved å behandle kreftceller med 5-aza-2’deoxycytidine (5-aza), en DNMT hemmer, vil DNAet demetyleres og miRNA uttrykkes
�Behandlet osteosarkom cellelinjer med 5-aza�Mengde uttrykt miRNA øker
� miRNA kan reguleres av metylering
miRNA og kreft – Epigenetikk og miRNA – Osteosarkom
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0
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60
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90
100
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72
Co
nfl
ue
ncy
(p
erc
en
t)
Hours (post-transfection)
miR-34c-5p
miR-335-5p
miR-486-5p
Negative control
Magne Skårn
Transfection with miRNAs affects cell proliferation
Bisulfite sequencing of osteosarcoma cell lines
methylated unmethylatedpartially methylated
Deeqa Ahmed
CpG sites in CpG island upstream of miRNA
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Verification of methylation pattern
in normal and patient samples?
qMSP – quantitative methylation specific RT-PCR
� miR-34b/c not methylated in patients
� miR-335 also methylated in normal samples ?
�miR-486 is cancer specific, an interesting biomarker
Deeqa Ahmed
450k methylation arrays
�Focused on wrong CpG island�Repeat qMSP of patient samples in the small CpG island
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En modell for miR-335 funksjon...
Acknowledgements
Dept. of Tumor Biology
• Anne-Mari Håkelien• Heidi M. Namløs• Magne Skårn• Kristine G. Harstad• Susanne Lorenz• Ola Myklebost
Dept of Cancer Prevention
• Deeqa Ahmed• Guro E. Lind
Broad Institute, MIT-Harvard
• Tarjei S. Mikkelsen
Genomics Core Facility
• Jan Christian Bryne• Jinchang Sun• Jonas Poulsen• Amin Madoui
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