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


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


also primary hMSC cultures and osteoblasts

Gene Expression Changes During

Osteogenic Differentiation


Undifferentiated

Dif

fere

nti

ate

d 1462 up-regulated

1695 down-regulated

Osteogenic Genes Mesenchymal Genes

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Epigenetic Regulation


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


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


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Histone Modifications Changes


Chromatin changes and gene expression


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Identification of Novel Transcriptional

Regulators of Osteogenesis


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


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


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


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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

10

20

30

40

50

60

70

80

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

leonardo a. meza-zepeda seminar august... · leonardo a. meza-zepeda dept. of tumorbiology the...

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