2. The role of epigenetic in the regulation of gene expression of cancer cells and viral diseases Presentation by : mostafa ghaderi Departemant of medical virology Tehran University,Iran

3. What is the epigenetics? Epigenetics are heritable changes that alter the expression of the gene without changing the DNA sequence. in other words: Epigenetics is the study of the inheritance of phonotypes that occur without a corresponding change in DNA sequance.

4. Chromatin Euchromatin Heterochromatin [Transcriptionally active] [Transcriptionally inactive]

6. Nucleosome with & without H1 + + + + + + - - - --- - - - - - - - - -

7. The importance (Meaning) of Histone modification (Histone code)

8. Genome alteration by DNA methylation. The methylation pattern is not random, instead being limited to the C in some copies of the sequences 5-CpG-3. Tow type of methylation activity have been distinguished. De novo methylation Dnmt1 Dnmt3a Dnmt3b Maintenance methylation Methylation is involved in genomic Imprinting, chromosomal stability and x inactivation DNA could be demethylated by: removing the methyl group, the base, or the nucleotide.

9.  DNA methylation is one of the layers of control of certain tissue-specific gene :  genes such as the MAGE genes, which are silent in almost all tissues except malignant tumors.  Genomic imprinting also requires DNA hypermethylation at one of the two parental alleles of a gene to ensure monoallelic expression.(such as Igf2 gene)  and a similar gene-dosage reduction is involved in X- chromosome inactivation in females.

10. DNA methylation refers to the transfer of a methyl (CH3 group) to one of the bases that constitute DNA. The reaction is catalyzed by a DNA methyltransferase (Mtase), and uses S- Adenosyl Methionine (SAM) as a methyl donor. In humans, normal DNA methylation is limited to the Cytosine base.

12. Aberrant Methylation DNA methylation has critical roles in the control of gene activity and the architecture of the nucleus of the cell. In humans, DNA methylation occurs in cytosines that precede guanines; these are called dinucleotide CpGs. CpG sites are not randomly distributed in the genome; instead, there are CpG-rich regions known as CpG islands, In humans, DNA methylation occurs in cytosines that precede guanines; these are called dinucleotide CpGs. CpG sites are not randomly distributed in the genome; instead, there are CpG-rich regions known as CpG islands, which span the 5′ end of the regulatory region of many genes. These islands are usually not methylated in normal cells.

13. ‘  Methylation of histones and DNA is connected.  HP1 is the key protein in forming mammalian hetrochromatin, and acts by binding to methylated histone H3.  Binding of HP1 to methylated H3 forms a trigger for silencing because further molecules of HP1aggregate on the nucleosome chain.  SUV39H1is a histone methyltransferase that acts on Lys 9 of histone H3.  Methyl-CpG binding proteins (MeCP2) as mediators of transcription repression :  MeCP2 interaction with co-repressor complex containing HDACs.

16. Acetylation Acetylating of histones is essential for gene expression and deacetylation is associate with repression of gene activity. Acetylating is reversible Enzymes that can acetylate histones are called histone acetyltransferases or HAT, the acetyl groups removed by histonedeacetylases or HDAC. TFIID and p300/CREB binding protein (CBP) have acetylase activity.

17. DNA Hypomethylation in Tumors Three mechanisms have been proposed to explain the contribution of DNA hypomethylation to the development of a cancer cell :  Generation of chromosomal instability, reactivation of transposable elements, and loss of imprinting. Undermethylation of DNA can favor mitotic recombination, leading to deletions and translocations, and it can also promote chromosomal rearrangements

18. DNA Hypermethylation in Tumors   Hypermethylation of the CpG islands in the promoter regions of tumor- suppressor genes is a major event in the origin of many cancers.   Hypermethylation of the CpG islands provides inactivation of the tumor-suppressor genes.(such as Rb,hMLH1, BRCA1)   Hypermethylation of the CpG islands in the promoter regions of tumor- suppressor genes in cancer cells is associated with a particular combination of histone markers: deacetylation of histones H3 and H4, loss of H3K4 trimethylation, and gain of H3K9 methylation and H3K27 trimethylation.

22. Main epigenetic alterations in cervical cancer

23. Epigenetic therapy agents inhibitors DNA methylation inhibitors DNA methylation inhibitors deoxycytidine analogs: 5-azacytidine, 5-aza-2-deoxycytidine Histone deacetylase inhibitors Histone deacetylase inhibitors Small molecular weight carboxilates: sodium butyrate, valproic acid

25. Epigenetics and Viruses  How can HSV undergo a lytic or latent infection In different cell types?  HSV DNA is not associate with histones inside the virion so, upon HSV entry into the host cell nucleus, it is likely that host cell mechanisms attempt to assemble chromatin on the viral DNA to silence the viral gene.

26. Chromatin and lytic infection v VP16 forms a complex with host cell factor (HCF) and localizes in the cell nucleus, where the VP16–HCF complex binds to the host transcription factor octamer-binding protein 1 (OCT1). vOCT1 binds to specific sites in the upstream regulatory sequences of IE genes, tethering the VP16–HCF complex to IE gene promoters and enabling the activator domain of VP16 to recruit transcription factors that stimulate IE gene transcription. vrecruited the chromatin-modifying co-activators CBP(cAMP response element (CREB)-binding protein) and p300, as well as components of the human orthologues of the yeast SwI/SNF ATP-dependent chromatin-remodeling complex (BRG1 and BRM) to viral IE gene promoters.

27. Chromatin and latent infection. Øthere are increased levels of acetylated H3 histone associated with the LAT promoter and enhancer compared with the ICP0 gene, which indicates that active chromatin was associated with the LAT gene only. ØHDAC inhibitor trichostatin A increases expression from the ICP0 gene promoter. Furthermore, treatment of latently infected mice with butyrate, another inhibitor of HDACs, causes acetylation of histones on lytic genes and reactivation of virus. ØOCT2 represses IE gene expression through its interaction with IE promoters that block OCT1 binding and activation

28.  Alternatively, LAT could enhance heterochromatin formation through a process that is similar to the mammalian X-inactivation process, in which a non-coding RNA, known as the X-inactivation short transcript (Xist), binds to one X chromosome and triggers chromosomal silencing.  Alternatively, the effect of LAT could be indirect.For example, the LAT transcripts might exert micro-RNA (miRNA) or antisense effects on ICP0 expression.  As discussed above, ICP0 increases active chromatin on viral lytic gene promoters, so reducing ICP0 expression could lead to increased heterochromatin

30. HIV and Epigenetic TTTTreatment of 4 individuals with a new HIV antiviral and valproic acid (VPA), an HDAC inhibitor, led to a decline in the number of infected resting CD4+ T cells. tttthere is compelling evidence that DNA-methylation influences HIV-1 replication. NNNNF-КB p50–HDAC1 complexes constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation. Suv39H1, HP1 and histone H3Lys9 trimethylation play a major role in chromatin-mediated repression of integrated HIV-1 gene expression

32.  in microglial cells, the co-repressor COUP-TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin- modifying complex and establishes a heterochromatic environment at the HIV-1 promoter.  CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV-1 promoter region. In addition,DNA-bound CTIP2 also associates with the histonemethyltransferase SUV39H1, which concomitant recruitment increases local histone H3 lysine 9 methylation. This allows of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV-1 silencing.

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