EpigeneticsBy

Anke van Eekelen, PhD

Telethon Institute for Child Health Research

100 Roberts RdSubiaco, WA 6008

9489 7886, ankev@ichr.uwa.edu.au

Cellular gene expression profiles underlie the establishment (development) and maintenance

of cellular identity

Epigenetics:

The events that change cellular expression profiles without changing the genetic code

I- chromatin modificationsII -microRNAs

I - chromatin modifications

the epigenotype of each gene is based on: histone/DNA modifications:

- histone acetylation

- DNA methylation

affect recognition and/or access of trans-acting factors to their binding sites

Altered gene activity

Histone acetylation/deacetylation

Acetylation of lysine

Histone

histone- histone-acetyltransferase deacetylases

Histone acetylation

Histones in transcriptionally active nucleosomes appear highly acetylated

Histone deacetylation

Gene silencing

DNA methylation

-methylation of genomic cytosine

Methyl transferase

methylated cytosine (methyl-CpG)

gene silencing

The epigenotype is somatically inheritable to maintain cellular identity upon differentiation (cellular memory)

Possible because methyl transferase prefers replicated DNA with methyl groups on one of the 2 strands

Methylation of new C’son new strand!

DNA methylation pattern dynamic during early development(crucial component of cell differentiation)

more stable in adult (differentiated) cells

II- microRNA

mRNA coding for proteins vs

Small nontranslated RNA:

- tRNAs : adapters in translation- rRNAs : building ribosomes- small nuclear RNAs: involved in mRNA splicing- small nucleolar RNAs: help rRNAs to make ribosomes

- small temporally regulated RNAs = stRNAs- small interfering RNAs = siRNAs

stRNA siRNA

21-25 nt long

mediate downregulationof gene expression

Target mRNAs after initiation target mRNA andof translation without affecting initiate degradationstability of mRNA of mRNA

* Known role in control of * mechanism exploreddevelopmental timing* Mechanism remains unclear * physiological role unclear

(Banerjee and Slack, BioEssays 24, 2002)

stRNA function: Heterochronic pathway of lin-4 and let-7 in C.Elegans

• Heterochronic genes control the timing of dev. programs and are temporal equivalents’ of homeotic spatial patterning genes

* Prococious mutants: cells inappropriately express later cell fates during early stages of development (lin-41 -/-)

* Retarded mutants: cells reiterate earlier stage fates instead of later wild-type fates (let-7 -/-)

stRNAsMechanism of action

Grosshans and Slack, JCB 156, 2002

Target mRNAs

Precursor RNAApprox 60-70 nt

Stem-loop structure

processing releasesmature stRNAfrom 5’arm

asymmetric cleavage

mRNA binding sites

stRNA ‘life cycle’

Grosshans and Slack, JCB 156, 2002

Unidentifiedpolymerase

tRNA is hypothesised totransport precursor RNAto cytoplasm

Pre-miRNA processing

Additional co-factorsAre believed to recognise the bulged-out nt ofmRNA/stRNA complex

miRNAs are NOT degraded products of longer RNAs !!!

- identification of miRNAs was based on RNA purification & size selection (verification on Northern blots)

- all miRNAs identified were encoded by genomic regions, for which no genes had been predicted

- surrounding genomic sequence of genes encoding for miRNAs predicted to form a stem-loop structure

- precursor miRNAs accumulate in Dicer -/- animals

Initial discovery of miRNA in C. Elegans, however..

…in 2001: 91 novel miRNAs were identified in 3 organisms* C.Elegans* Drosophila* Humans (2005: approx 200!)(*zebrafish)

…some miRNAs seem preserved across phyla (perfect homologues)

…also conservation of the polymerase II-like protein Dicer (drc-1 in C. Elegans; Dicer in Drosophila; helicoise-MOI in humans)

Posttranscriptional regulatory mechanism of gene expression possibly Acting in a wide variety of other organisms than C. Elegans

stRNA siRNA

Binding sites for stRNAs in 3’ UTR of mRNA binding sites for siRNAs anywhere on mRNA

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RNA interference approach for posttranscriptional gene silencing

RISC: RNA-induced silencing complex

Step 1: dsRNA cleavage by Dicer

Step 2: recruitment of siRNA & RNAi factors and formation of RISC

Step 3: siRNA-unwinding and RISC activation

Step 4: mRNA targeting and degradation

Various strategies of RNAi in Mammalian cells

Long dsRNA microinjectionuse of internal Dicer

Synthetic siRNA transfectionuse of internal Dicer

Diced siRNA transfectionimmediate RISC formation & activation

Plasmid-based shRNA vector transfectioninternal nuclear transcription and processing

Virus-based shRNA vector transfectioninternal nuclear transcription and processing

References

H. Grosshans and F. J. Slack (2002) Micro-RNAs: small is plentiful. JCB minireview, 156: pp 17-21

D. Banerjee and F Slack (2002) Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression. BioEssays 24: pp 119-129.

V.N. Kim (2003) RNA interference in functional genomics and medicine. J Korean Med Sci review 18: pp 309-318 .