Gene Silencing Arlés Urrutia Biomedicine 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) 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 ExpressionDNA repairDNA ReplicationChromosome Condensation Methylation H3K4me, H3K36me (3)  RNApol II H4K20  Crb2 H4K20me, 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) HISTONE MODIFICATION

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

15 February 2001

CHROMATIN IMMUNOPRECIPITATION ASSAY

INHERITANCE & LIFE TIME Chromosome 15 (q11-13) are deleted or unexpressed MOTHERFATHER Angelman Syndrome. neuro-genetic disorder Happy demanor Prade-Willi Syndrome Maternal Allele Silenced. Cognitive, Breathing and feeding dificulties.

INHERITANCE & LIFE TIME 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

DISEASES Epigenetic 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? 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). INHERITANCE & LIFE TIME

Spain Research. CNIO. The Experts

Williamson S. Christodoulou, J. (2006) Rett Syndrome: new clinical and molecular insights. Eur J Hum Genet. 14, 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 – Robertson, K. (2005) DNA methylation and human disease. Nature Reviews,Genetics. 6:597. Jones, P. Baylin, S. (2007) The Epigenomics of Cancer. Cell. 128: 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: Cullum, R. Alder, O. Hoodless, P. (2011) The Next generation: Using new sequencing technologies to analyse gene regulation. Respirology. 16: REFERENCES