Gene Silencing

Gene Silencing

Arlés UrrutiaBiomedicine and Biotechnology Institute.

Autonomous University of Barcelona. (IBB-UAB)Neurochemistry Lab.

How do the many cell types of the body maintain drastically different gene expression patterns

while sharing exactly the same DNA?

Evolutionary SNP´s – CNV´s

Protein Expression

Biochemical Activity

• DNA methylation– Methyl-CpG-binding proteins: MeCP2.– De novo methyltransferase: Dnmt1.– mSinA3 – HDAC Complex.

• Histone Modification.– Acetilation & Methylation

• Chromatin Immunoprecipitation Assay• Inheritance & Life Time• Diseases & Therapies

CONTENT

Schematic diagram of the molecular mechanism of methylase inheritance and transcriptional repression. HDAC1/2-mSin3A-MeCP2 Complex organized chromatin integrity. (Kimura, 2003)

Hemimethylated

mSin3AHDAC1/2

HDAC1/2

HDAC1/2

HDAC1/2 MeCP2

mSin3A

MeCP2

mSin3A

MeCP2

Hemimethylated

mSin3AHDAC1/2

HDAC1/2

HDAC1/2

HDAC1/2 MeCP2

mSin3A

MeCP2

mSin3A

MeCP2

DNA METHYLATION

Recruitment of Proteins to Histones. B. Proteins found that associated preferentially with modified versions of histones H3 and histone H4. (Kouzarides, 2007)

Carpenter (Abcam), 2005

HISTONE MODIFICATION

Gene Expression DNA repair DNA Replication Chromosome Condensation

Methylation

H3K4me, H3K36me (3) RNApol II

H4K20 Crb2H4K20me, H3K79me p53BP1

H3K9me H3S10ph

Acetylation

H3K56Ac (+)H4K16Ac (-)

H3K56Ac, H4K12Ac

H4K12Ac, H4K8Ac, H4K5Ac HBO1 - Pre-replicative complex

H4K16Ac

(+) , (-) : activation or inhibition of the process. throught the interaction with.(Fuks, 2005)



Slide courtesy of Trevor Archer adapted from Jenuwein & Allis, Science 2001

HISTONE CODE

HAT HDAC

15 February 2001

CHROMATIN IMMUNOPRECIPITATION ASSAY

INHERITANCE & LIFE TIME

Chromosome 15 (q11-13) are deleted or unexpressed

MOTHER FATHERAngelman Syndrome. neuro-genetic disorder

Happy demanor

Prade-Willi SyndromeMaternal Allele Silenced. Cognitive, Breathing and

feeding dificulties.


Mapping chromosomal regions with differential DNA methylation in MZ twins by using comparative genomic hybridization for methylated DNA. Presence of green and red signals that indicate hypermethylation and hypomethylation events, whereas the 3-year-old twins have a very similar distribution of DNA methylation indicated by the presence of the yellow color.

Fraga et al, 2005

DISEASESEpigenetic therapy with DNA methylation inhibitors (DNMTi) and HDAC inhibitors (HDACi). Combination therapies are likely to gain traction in the future because of the inherent self – reinforcing nature of silencing mechanisms.

Future breakthroughs could come from the use to epigenetic drugs to activate miRNAs or use of drugs to target cancer stem cells after tumor debulking by estándar chemotherapy. (Jones & Baylin, 2007)

Changing the view of what Inheritance is.• Our parents life is affecting us directly, during the eggs and

embrio developments. Do the genes have memory?

• 30.000 genes is not enough for this ammount of differences between individuals, even identical twins.

• The apereance of Beckwith-Wiedemann syndrome, (BWS) is 3/65 by In Vitro Fertilization in Victoria, AU. Could this procedure trigger epigenetics switches for disseased?

• Is the imprinting an adaptation from the parents to the next generation? The Overkalix case, in Sweden (food availability and longevity).


Spain Research . CNIO.

The Experts

• Williamson S. Christodoulou, J. (2006) Rett Syndrome: new clinical and molecular insights. Eur J Hum Genet. 14, 896-903.

• Fuks, F. (2005) DNA methylation and histone modifications: teaming up to silence genes. Curr Opi in Genet & Develop. 15:1-6.

• Kimura, H. Shiota, K. (2003) Methyl-CpG-binding protein, MeCP2, is target Molecule for Maintenance DNA Methyltransferase, Dnmt1. J.Biol. Chem. 278:4806 – 4812.

• Robertson, K. (2005) DNA methylation and human disease. Nature Reviews,Genetics. 6:597.

• Jones, P. Baylin, S. (2007) The Epigenomics of Cancer. Cell. 128:683-692.• Nan, X. Ng, HH. Johnson, C. Laherty, C. Turner, B. Eisenman, R. Bird, A. (1998)

Transcriptional repression by methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature Letters. 393:386-389.

• Cullum, R. Alder, O. Hoodless, P. (2011) The Next generation: Using new sequencing technologies to analyse gene regulation. Respirology. 16:210-222.

REFERENCES

Gene Silencing

Documents

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