Mechanism of Epigenetics and it’s Role in the Gene Regulation

1. Mechanism of Epigenetics and it’s Role in the Gene Regulation

2. Group B • SaniaSafdar Butt • Maheen Nawaz • WajeehaJaved Khan • Deeba Hassan • TunjeenaRehan

3. EPIGENETICS: AN INTRODUCTIONby SaniaSafdar Butt

4. Epigenetics ; The Science of Change ‘ Genes load the gun and the environment pulls the trigger’ Bruce Lipton

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6. “Epigenetics” literal meaning • Epigenetics means "above" or "on top of" genetics. • It refers to external modifications to DNA that turn genes "on" or "off." • These modifications do not change the DNA sequence, but instead, they affect how cells "read" genes. • “in addition to changes in genetic sequence’’.

7. Nature VS Nurture

9. Epigenetics

10. History • In the early 1940s, Dr. Waddington, an embryologist, put forth a radical idea for its era • Originally used to denote the poorly understood processes by which a fertilized zygote developed into a mature, complex organism. • The definition was changed to focus on ways in which heritable traits can be associated not with changes in nucleotide sequence.

11. Epigenetics associated with: • Chemical modifications of DNA • The structural and regulatory proteins bound to it • Altering the gene activity without changing the DNA sequence • Modifications that can transfer to the daughter cells.

12. Epigenetic Marks • Epigenetics is essentially additional information layered on top of the sequence of letters (strings of molecules called A, C, G, and T) that makes up DNA. • DNA can be tagged with tiny molecules, the methyl groups that stick to Cysteine residues. • Tags can be added to proteins called histones that are closely associated with DNA

13. Epigenetic Inheritance • Environmental factors experienced by adult mice can be passed on to their offspring via epigenetic mechanisms. • The best example is a gene called “agouti”: • Methylated in normal brown mice. • Unmethylated genecontaining mice are yellow and obese (despite being genetically essentially identical to their skinny brown relatives). • Altering the pregnant mother’s diet can modify the ratio of brown to yellow offspring • Folic acid results in more brown pups • BPA (Bisphenol A) results in more yellow pups.

14. Obese and Healthy Mice

15. Epigenetic Inheritance • It is possible to pass down epigenetic changes to future generations if the changes occur in sperm or egg cells. • Most epigenetic changes that occur in sperm and egg cells get erased when the two combine to form a fertilized egg, in a process called "reprogramming”. • Scientists think some of the epigenetic changes in parents' sperm and egg cells may avoid the reprogramming process, and make it through to the next generation.

16. Examples Epigenetics is the reason why a skin cell looks different from a brain cell or a muscle cell. All three cells contain the same DNA, but their genes are expressed differently (turned "on" or "off"), which creates the different cell types.

17. References • Felsenfeld, G. (2014). A brief history of epigenetics. Cold Spring Harbor perspectives in biology, 6(1), a018200. • Weinhold, B. (2006). Epigenetics: the science of change. Environmental Health Perspectives, 114(3), A160. • Jones, P. A., & Takai, D. (2001). The role of DNA methylation in mammalian epigenetics. Science, 293(5532), 1068-1070.

18. MECHANISM OF EPIGENETICS by Deeba Hassan

19. Epigenetic refer to all mitotically and meiotically heritable changes in gene expression which are not coded in DNA sequence. They are not decided by the changes in DNA. Epigenetic gene activities become stable and can be inherited through the generations very rarely.

20. Epigenetics acts mainly through four different mechanism DNA methylation Chromatin remodelling Histone modification Expression of regulatory RNAs

21. DNA Methylation A. CYTOSINE METHYLATION • Occurs on 5’ Position of Cytosine in the context of the dinucleotide sequence CpG. • Majority (75%) of all CpG dinucleotides in the mammalian genome are methylated.

22. DNA Methylation B. METHYLATION AT PROMOTER SITE In principal: • Methylation of CpG (e.g. within promoter region) • Increase: Reduced transcriptional activity • Decrease: Increased transcriptional activity Promoter region

23. Natural Roles of DNA Methylation in Mammalian System Imprinting X chromosome inactivation Heterochromatin maintenance Developmental controls Tissue specific expression controls Silencing of parasitic DNA elements Transcriptional regulation Genomic stability

24. CHROMATIN REMODELLING It includes the shifting of nucleosome core. This process is known as nucleosome sliding. The shift may result from disassembling and reassembling of nucleosome core. This process is major factor for controlling the gene expression by induction and repression.

25. HISTONE MODIFICATIONS • There are different histone modifications which are typically conserved over evolutionary process. • These modifications occur both at coding and non coding sequences of genome.

27. HISTONE MODIFICATIONS

28. REGULATORY RNAs A non-coding RNA (ncRNA) is a functional RNA molecule that is transcribed from DNA but not translated into proteins. Epigenetic related ncRNAs include miRNA, siRNA, and lncRNA. Play a role in heterochromatin formation, histone modification, DNA methylation targeting, and gene silencing.

29. EPIGENETICS IN GENE REGULATIONby Maheen Nawaz

30. MECHANISM OF METHYLATION • The methyl group is usually covalently added to 5-carbon of the cytosine ring resulting in 5-methylcytosine (5-mC). • In human DNA, 5-mC is found in approximately 1.5% of genomic DNA. • In somatic cells, 5-mC occurs almost exclusively at CpG sites. • In embryonic stem cells, substantial amount of 5-mC is also found in non-CpG regions. • In genomic DNA, • CpG sites (mostly) are heavily methylated • CpG islands (sites of CpG clusters) in germ-line tissues and located near promoters of normal somatic cells, remain unmethylated

31. When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed/turned off. • It is observed that the CpG islands in the promoters of housekeeping genes (genes expressed in most cells) are mainly unmethylated, and that methylation of CpG islands in cancer cells often leads to silencing of gene expression, led to the hypothesis that DNA methylation plays an important role in regulating gene expression

32. Fig: Percentage of 5-fC in genomic DNA of different tissues (5-mC is hydrolysed to 5-fC)

33. STEPS OF METHYLATION The addition of methyl groups is controlled at different levels in cells by a family of enzymes, DNA methyltransferases (DNMTs) • DNMT1 maintains established DNA methylation • DNMT3a and DMNT3b establish new methylation patterns • DNMT2 and DNMT3L have more specialized but related functions Apart from DNMTs, other methylating factors include: • Histone modifications (acetylation and methylation) • Nucleosome positioning (by ATP-dependent chromatin remodeling enzymes) DNA methyltransferases(DNMTs) interact with histone deacetylases to repress transcription

34. MECHANISM OF EXPRESSION INHIBITION • For gene transcription to occur, the gene promoter should be readily accessible to transcription factors and other regulatory units. • The methyl groups project into the major groove of DNA altering the chromatin structure directly (or indirectly by altering histones) and restricting the access of transcription factors to the gene promoter • For example, methylated CpGs attract methyl-CpG-binding domain proteins that recruit ‘repressor complexes’, resulting in histone modification. The modification of histones by the recruitment of repressor complexes leads to a more condensed chromatin structure (heterochromatin) as opposed to an open and active chromatin structure (euchromatin) required for transcription.

35. DNA methylation regulating gene expression: • The CpG island promoter is unmethylated and allows binding of transcription factors, which is required for transcription initiation • The CpG island promoter methylation prevents binding of transcription factors and results in gene silencing.

36. DNA methylation and genomic imprinting • Imprinted genes are selectively expressed depending on the parental chromosome of origin i.e., only one allele of the gene is expressed. Only 80 human genes are imprinted. Example: • For Insulin-like growth factor 2 (IGF2), only paternal allele is expressed • For Closely linked H19 gene, only maternal allele is expressed • Imprinted genes play a critical role in fetal and neuro-development. Thus, two copies of the paternal genome (and no maternal contribution) result in a complete molar pregnancy despite the correct number of genes being present.

37. DEMETHYLATION • The removal of a methyl group, DNA demethylation is as important as DNA methylation. • It is necessary for epigenetic reprogramming of genes and is also directly involved in many important disease mechanisms such as tumor progression. • Demethylationof DNA can either be passive or active, or a combination of both: • Passive DNA demethylation: occurs on newly synthesized DNA strands via DNMT1 during replication • Active DNA demethylation: removal of 5-mC via modification of cytosine by enzymes. • The TET(demethylating) proteins promote DNA demethylationby: • Binding to CpG rich regions to prevent unwanted DNA methyltransferaseactivity • Sequentially converting 5-mC to 5-caC (5-carboxylcytosine) through hydroxylase activity. • These proteins function in transcriptional activation and repression (TET1), tumor suppression (TET2), and DNA methylation reprogramming processes (TET3).

38. REFERENCES: • Robertson, K.D.DNA methylation and chromatin - unraveling the tangled web. Oncogene. 21(35), 5361-79 (2002) • Jones, P.A. & Takai, D. The Role of DNA Methylation in Mammalian Epigenetics. Science  293(5532), 1068-1070 (2001). • Grewal, S. I. S. and Moazed, D. Heterochromatin and Epigenetic Control of Gene Expression. Science 301(5634) , pp. 798-802 (2003). • Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425-432 (2007). • Jaenish, R. and Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental stimuli. Nature genetics 33, 245-254 (2003). • El-Osta, A. andWolffe, A. P. DNA methylation and histone deacetylation in the control of gene expression: basic biochemistry to human development and disease. Gene Expressions 9(1-2), 63-75(2000). • Newell-Price, J.,Clark, A.J., andKing, P. DNA methylation and silencing of gene expression.Trends in Endocrinology and Metabolism 11(4), 142-8 (2000). • Allis, C.D., Jenuwein, T., Reinberg, D., and Caparros, M.L., eds. (2007). Epigenetics (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

39. EPIGENETIC DISEASESbyWajeehaJaved Khan

40. An accumulation of genetic and epigenetic errors can transform a normal cell into an invasive or metastatic tumor cell. • DNA methylation patterns may cause abnormal expression of cancer-associated genes. • Subsequently, epigenetic changes can be used as biomarkers for the molecular diagnosis of early cancer.

41. Cancer • Cancer was the first human disease to be linked to epigenetics. • Studies performed by Feinberg and Vogelstein in 1983, using primary human tumor tissues, found that genes of colorectal cancer cells were substantially hypomethylated compared with normal tissues. • DNA Hypomethylation can activate oncogenes and initiate chromosome instability. • DNA Hypermethylation initiates silencing of tumor suppressor genes.

42. Mental Retardation Disorders. • Epigenetic changes are also linked to several disorders that result in intellectual disabilities such as : • ATR-X • Fragile X • Prader-Willi • Angelmansyndromes.

43. The imprint disorders Prader-Willi syndrome and Angelman syndrome, there is a genetic deletion in chromosome 15 in a majority of patients. • The same gene on the corresponding chromosome cannot compensate for the deletion because it has been turned off by methylation, an epigenetic modification.  • Genetic deletions inherited from the father result in Prader-Willi syndrome, and those inherited from the mother, Angelman syndrome.

44. Immunity & Related Disorders • There are several pieces of evidence showing that loss of epigenetic control over complex immune processes contributes to autoimmune disease. • Abnormal DNA methylation has been observed in patients with lupus whose T cells exhibit decreased DNA methyl transferase activity and hypomethylated DNA. • Disregulationof this pathway apparently leads to overexpression of methylation-sensitive genes such as the leukocyte function-associated factor (LFA1), which causes lupus-like autoimmunity.

45. Why Should You Care?

46. Epigenetics across the human lifespan • In differentiated cells, signals fine-tune cell functions through changes in gene expression across the lifespan. • A flexible epigenome allows us to adjust to changes in the world around us, and to “learn” from our experiences • The “software” of the genome and directs embryogenesis and development,

47. Epigenetics across the human lifespan • Unique sets of genes are induced or silenced epigenetically during different stages of life and these are responsible for the development and maturation of the individual through orchestrated events in combination with input from the environment. • An imbalance in the regulation process, and might have a life-long effect on the individual

48. From the periconceptional environment to birth • For most genes, total reprogramming is necessary very soon after conception in order to start with an epigenetic “clean slate” • In the fertilized egg: global DNA demethylation is followed by remethylation to reprogram the maternal and paternal genomes for efficient gene expression regulation.

49. From the periconceptional environment to birth • The fetalepigenome is most susceptible during this developmental period to epigenetic modifiers in the maternal environment. An error during such a crucial time might lead to an abnormal phenotypic outcome in the offspring.

50. Effect of Assisted reproductive technologies on epigenetics • Assisted reproductive technologies (ARTs) like IntraCytoplasmic Sperm Injection (ICSI) and in vitro feritlization (IVF), which are used in case of male or female sub-fertility, cause epigenetic changes in offspring such that there is differential allelic expression in the early embryo (Kohda and Ishino, 2013). • While comparing the two ARTs, ICSI, and IVF, both result in epigenetic errors owing to aberrant DNA methylation, but neither one has an increased effect as compared to the other (Santos et al., 2010).