Epigenetic Mechanisms PDF

EPIGENETIC MECHANISMS ASSIST. PROF.DR IDIL ASLAN UNIVERSITY OF KYRENIA MEDICAL SCHOOL INTRODUCTION The term 'epigenetics' was initially proposed by Conrad Waddington in 1942 In Ancient Greek, the prefix 'epi' means 'upon,' 'over,' or 'beyond.' Therefore, 'epigenetic' implies 'a...

EPIGENETIC MECHANISMS ASSIST. PROF.DR IDIL ASLAN UNIVERSITY OF KYRENIA MEDICAL SCHOOL INTRODUCTION The term 'epigenetics' was initially proposed by Conrad Waddington in 1942 In Ancient Greek, the prefix 'epi' means 'upon,' 'over,' or 'beyond.' Therefore, 'epigenetic' implies 'above genetics' and examines changes in gene expression. Epigenetic regulation refers to changes in gene expression that can be passed on through mitotic and/or meiotic cell division without altering the DNA's nucleotide structure. Genome distinguishes one individual from another, while epigenome distinguishes one cell from another.  Various tissues and organs in humans and animals are comprised of numerous cells. While the DNA sequence of each cell remains the same The timing, location, and duration of the replication, transcription, and translation processes during DNA-to- protein synthesis can vary among cells. For instance, tissues of organs like the brain, liver, or heart, despite having the same DNA sequence, exhibit differences in shape and function. This discrepancy arises from certain genes being active or silent based on the cell type. This cellular differentiation can serve as an example of epigenetic regulation. Epigenetic Mechanisms Epigenetic mechanisms are divided into two categories: Mechanisms that directly control and influence gene expression (Transcriptional) Mechanisms that indirectly control and influence gene expression (Post-transcriptional, mRNA silencing) I- Mechanisms Directly Controlling Gene Expression 1- Modifications at the DNA Level - DNA Methylation 2- Modifications at the Chromatin Level A- Histone Modifications (Covalent Modifications): - Histone Acetylation - Histone Methylation - Histone Deimination - Glycosylation (β-acetylglucosaminylation) - ADP Ribosylation - Histone Phosphorylation - Histone Ubiquitination - Histone Sumoylation - Nitrosylation - Biotinylation  B- Non-Covalent Modifications: - Histone Exchange - Interaction with Non-Coding mRNA (miRNA, siRNA) - Histone Deposition - Interaction with Other Agents (e.g., viruses) - Chromatin Repair II- Mechanisms Indirectly Controlling Gene Expression Post-transcriptional mechanisms primarily involve the inhibition of protein synthesis through the impact of non-coding RNA on coding mRNA. I-Chromatin Modifications: A- Histone Modifications (Covalent Modifications): Histone modifications alter chromatin structure, creating recognition sites for transcription regulatory proteins; these changes play a regulatory role in gene expression. How these modifications affect chromatin structure has been revealed through high-resolution X-ray crystallography imaging of nucleosomes. Most histone modifications are reversible, and the level of modification is closely correlated with transcription levels. These modifications can occur individually or in various combinations, conferring or altering specific meanings to chromatin. As a result, histone-DNA and histone-histone interactions are influenced, enabling the regulation of numerous biological processes such as DNA packaging, replication, repair, and the control of gene expression. 1. Histone Acetylation When a negatively (-) charged acetyl group attaches to the amino terminus of a histone protein, the positively (+) charged lysine amino acid partially loses its charge, converting into neutral amide bonds. This weakens the electrostatic interaction between histones and DNA, leading to a relaxation of the chromatin structure. As a result, transcription factors can more easily access the promoter regions of genes, exposing the DNA to enzymes like RNA polymerase and facilitating gene transcription. Histone acetylation is regulated by two enzymes: histone acetyltransferase (HAT) and histone deacetylase (HDAC). (Further studies have revealed that a subtype of HDAC plays a role in regulating embryonic stem cell differentiation.) Acetylation is a reversible process. When the acetyl group is removed from acetylated lysine amino acids, chromatin condenses again, suppressing transcription. Acetylation of histones in a specific chromatin region indicates transcriptional activity in that region, whereas deacetylation signifies transcriptional repression. 2- Histone Methylation: The transfer of methyl groups from S-adenosyl methionine (SAM) to histones is catalyzed by an enzyme called histone methyltransferase (HMT). Unlike histone acetylation or phosphorylation, histone methylation does not alter the charge of histones. HMT enzymes target arginine or lysine residues on histones. Arginine residues can be mono- or dimethylated, often resulting in transcriptional activation. Lysine methylation, on the other hand, can lead to either activation or repression of transcription, depending on the specific residue that is methylated. Until 2002, histone methylation was believed to be stable and irreversible. However, in 2004, the discovery of specific demethylases (HDT) confirmed that this process is reversible. Methylated histones at specific amino acid residues can exert epigenetic effects, either activating or repressing gene expression. 3- Histone Deimination: Histone deimination involves the conversion of arginine residues to citrulline. This reaction is catalyzed by the enzyme "peptidyl deiminase" (PAD). As a result of this conversion, the positive charge on arginine is neutralized, leading to chromatin relaxation. This relaxation facilitates the access of transcription factors to DNA, promoting gene expression. However, in certain contexts, deimination can contribute to chromatin compaction, thereby repressing transcription. 4- Histone Ubiquitination Ubiquitination induces larger molecular changes within the structure of histones. Ubiquitin, as is well known, is a 76-amino acid polypeptide involved in the formation of proteins' three-dimensional structures. It is attached to lysine residues on histones through the action of an enzyme complex composed of three enzymes:(E1 - activating enzyme, E2 - conjugating enzyme, and E3 - ligating enzyme). This process weakens the bonds between histones and DNA, resulting in chromatin relaxation and the activation of transcription. 5- Histone Sumoylation: Sumoylation is a modification associated with ubiquitination It involves the attachment of small ubiquitin-like modifier (SUMO) molecules to the lysine residues of histones through E1, E2, and E3 enzymes. Its function is the opposite of acetylation and ubiquitination, meaning it leads to transcriptional repression. 6-Histone Phosphorylation Phosphates bind to histone deacetylases through serine, threonine, and tyrosine residues. Phosphorylation is the reversible attachment of phosphate groups to histone deacetylases by protein kinases (PK) and protein phosphatases (PP)." B- Non-Covalent Modifications: These include histone exchanges, histone incorporations, chromatin repair, intrachromosomal interactions, and interactions with noncoding RNA. Among these small RNA molecules, known as RNA (non-coding RNA), which have been shown to play a role particularly in epigenetic processes, miRNA (micro RNA) and siRNA (small- interfering RNA) lead to post-transcriptional and post- translational silencing The Control of Gene Expression by RNA Only about 3% of the mammalian genome consists of protein-coding messenger RNAs (mRNA). The effects of the remaining 97% have not been fully elucidated. Within this portion, non-coding RNAs (ncRNAs) are synthesized, which do not encode proteins. Some of these ncRNAs are reported to be involved in epigenetic processes.  Some ncRNAs hinder the transcription stage in the nucleus, while others in the cytoplasm are known to suppress gene expression by cleaving or degrading mRNAs or halting translation. Additionally, ncRNAs are believed to play a role in initiating histone modifications and DNA methylation, contributing to the formation of heterochromatin regions, thereby facilitating DNA silencing.  The pathways used by the cell to suppress certain genes, known as RNA interference (RNAi) pathways, primarily involve small interfering RNA (siRNA) and microRNA (miRNA) particles. These RNAs interact with mRNA within a complex rich in RNA and RNA-binding proteins called the RNA-induced silencing complex (RISC). siRNA perfectly matches the target mRNA sequence, leading to the cleavage of the mRNA molecule at the matching regions by endonucleases  Unlike siRNA, miRNA generally cannot cleave mRNA because it doesn’t exhibit perfect matching with mRNA. However, it can suppress translation by preventing the binding of initiation factors to mRNA or by causing the dissociation of mRNA from ribosomes during ongoing translation. Nonetheless, some miRNAs, similar to siRNA, can exhibit perfect complementarity with mRNA and can degrade mRNA, thus inhibiting gene expression II-DNA MODIFICATION - DNA Methylation: One of the most studied, known, and functionally important modifications at the DNA level is DNA methylation. Generally, DNA methylation occurs when a methyl group (CH3) attaches to cytosine in CpG (Cytosine, Phosphate, Guanine) dinucleotides, which are frequently found in CpG islands in vertebrates. CpG islands in housekeeping and regulatory genes, which continuously need expression for an organism's development and survival, are typically resistant to DNA methylation. Conversely, in heterochromatin regions like repeat sequences and transposons, CpG sequences exhibit high levels of DNA methylation. This higher methylation rate helps prevent unwanted tissue- specific transcriptional activity. Around 70% of CG nucleotides in the genome are methylated. It facilitates gene silencing. DNA Methyltransferases: The enzyme responsible for adding a methyl group to the 5th carbon of cytosine in DNA is called “DNA methyltransferase” When both strands are methylated, it's termed as full methylation, while methylation on one strand is referred to as hemimethylation. Methylation occurs at CpG islands, which are typically around 1000-2000 base pairs in length. DNA methyltransferases are generally divided into two main groups: De Novo DNA Methyltransferases: These enzymes are responsible for creating new methylation in DNA. Particularly during cell division and early stages of development, they initiate the epigenetic modification of DNA by adding methyl groups to regions of DNA that have not previously undergone methylation. Maintenance DNA Methyltransferases: These enzymes are necessary to preserve and replicate existing methylation patterns. During DNA replication, these enzymes methylate newly synthesized DNA strands to maintain the previous methylation patterns. DNMT1: Facilitates maintenance methylation, aiding in the preservation of methylation patterns during DNA replication. DNMT2: Has weak DNA methyltransferase activity and behaves like a tRNA methyltransferase. DNMT3A and DNMT3B: Perform De Novo methylation, initiating methylation by adding methyl groups to new DNA regions. They are heavily expressed in embryonic cells. Differences Between Epigenetic Changes and Genetic Changes: Persistence: Genetic changes are permanent and can be passed from one generation to the next. Epigenetic changes, on the other hand, are usually reversible or modifiable during cell division. However, some epigenetic marks can be inherited across generations. DNA Sequence: Genetic changes involve a direct alteration in the DNA sequence. Epigenetic changes, however, do not modify the DNA sequence; they only affect gene expression. Mechanism of Action: Genetic changes typically involve modifying or affecting the coding of a specific gene. Epigenetic changes regulate how much or when a gene is expressed. Epigenetic Mechanisms and Cancer Epigenetic modifications play a crucial role in cancer development. Key mechanisms include: 1- Silencing of Tumor Suppressor Genes: Hypermethylation of tumor suppressor genes prevents their expression, leading to uncontrolled cell division. 2- Activation of Oncogenes: DNA methylation and histone modifications can activate oncogenes, which promote cancer formation. 3- Genomic Instability: Epigenetic mechanisms can impair DNA damage repair genes, resulting in genomic instability and cancer. 4- Epigenetic Drug Resistance: Certain epigenetic changes may confer resistance to cancer treatments. Treatment and Epigenetic Regulation The reversible nature of epigenetic mechanisms has sparked interest in epigenetic-based therapies. Treatments for cancer that target epigenetic changes include: - DNA Methylation Inhibitors Examples: DNA methyltransferase enzymes to reactivate tumor suppressor genes. - Histone Deacetylase (HDAC) Inhibitors: Target histone deacetylases to regulate gene expression. - miRNA Therapies: Aim to correct abnormal miRNA expression. Epigenetic Events: Through epigenetic mechanisms, regulation occurs in the expression or suppression of genetic information present in DNA. These mechanisms govern epigenetic events such as genomic imprinting, gametogenesis (the process of gamete formation), gene activation and inactivation in embryos, X chromosome inactivation, cellular rejuvenation in adults, and other similar processes." Dosage Compensation (X Chromosome Inactivation): Dosage compensation aims to maintain an equal dosage of many genes on the X chromosome between the two sexes. It is a process found in female mammals, particularly in humans and other mammalian species. In this process, one of the X chromosomes in female individuals becomes inactive to maintain genetic balance. In mammals, this process becomes active during embryonic development. In female mammals, the equalization of expression occurs by silencing one of the X chromosomes through a process called X chromosome inactivation. During this process, one X chromosome is randomly selected and rendered inactive. X Chromosome Inactivation In 1949, Murry Barr and Ewart Bertam observed a highly condensed mass in the nuclei of somatic cells during the interphase in female cats. This structure was not observed in male cats. This structure was named the Barr Body. In 1960, it was confirmed that this structure was a compressed X chromosome. During and After Inactivation: Chromosomal DNA condenses, forming the Barr Body. Due to this condensation, many genes on the X chromosome cannot express themselves. During cell division, the inactive X chromosome undergoes replication, and after replication, both copies remain condensed and inactive. Consequently, in all subsequent cell divisions, the inactive X chromosome will be passed on to all daughter cells. X Inactivation Center and Xist Gene The genetic control of inactivation is not fully understood at the molecular level. A region on the X chromosome is believed to play a role, known as the X inactivation center (Xic). If Xic is missing on the chromosome, inactivation does not occur. In humans, having two fully active X chromosomes is lethal during the embryonic period. Xist There's a specific gene responsible for initiating and regulating X inactivation, and that's the Xist gene. This gene resides within the X inactivation center (XIC) and plays a critical role in initiating the process of inactivation. The Xist gene produces a long non-coding RNA responsible for the inactivation of the X chromosome. This RNA molecule gathers on the inactive X chromosome, leading to the silencing of genes in that region. Thus, the Xist gene functions as a key regulator in silencing the inactive X chromosome. Xist and Tsix The role of Xist RNA is to coat the X chromosome and render it inactive. Once Xist RNA coats the chromosome, proteins interact with this RNA, facilitating the condensation of the chromosome and the formation of the Barr Body. Within the XIC region, there's a second gene called Tsix, which functions to prevent X inactivation. These two genes overlap and transcribe in opposite directions. If there's a mutation in the Tsix gene of an X chromosome, preferably that X chromosome will be rendered inactive The three phases of inactivation 1- Initiation: This phase begins with the activation of the Xist gene. Initially, the Xist gene, located in the X inactivation center (XIC), becomes active. Xist RNA is produced, coating the X chromosome to be inactivated. This process marks the first step towards silencing the X chromosome. 2- Spreading: Xist RNA starts to spread across the chromosome, interacting with specific proteins, leading to the condensation of the chromosome and the formation of a condensed structure known as the Barr Body. This phase involves the expansion of inactivation and the physical silencing of the X chromosome. 3- Maintenance: This phase focuses on preserving the inactive state of the X chromosome. Once the Barr Body is formed, this inactive status is maintained during cell division and passed on to new cells, ensuring the continuity of the X chromosome's inactive state. Genetic Imprinting: 'Imprinting' is used to describe the suppression of an expressible gene. The effect resulting from the suppression of a gene can be either positive or negative. Gene suppression involves the epigenetic mechanisms that suppress gene expression without changing the DNA sequence. Therefore, gene suppression (imprinting) is a reversible gene inactivation, not a mutation.  In genomic imprinting, certain genes are silenced based on parental origin. Genomic imprinting was first demonstrated through nuclear transfer studies in 1984, revealing functional inequality between maternal and paternal genomes in certain regions, resulting in differences in gene locus activity based on parental origin  This mechanism suppresses one of the maternal or paternal alleles, allowing only monoallelic expression, resulting in differential expression based on the parental origin of the genes being transmitted. Imprinted genes, particularly those with dense CpG islands, display distinct methylation patterns specific to different alleles. The expression of these genes is regulated by imprinting control regions (ICRs). This often leads to non-Mendelian inheritance as it enables the organism to distinguish between maternal and paternal alleles Imprinting and Human Diseases: The most characteristic examples within this group of disorders are Prader-Willi and Angelman syndromes. There exists a series of genes on chromosome 15 that exhibit paternal and maternal monoallelic expression. Imprinting control regions (ICRs) regulate the expression of these genes, controlling the mechanism of different genes found on paternal and maternal chromosomes with their bipartite structure. Deletion of the Small nuclear ribonucleoprotein associated protein N gene's promoter and ICR leads to Prader-Willi syndrome on the paternal chromosome. The deletion renders the imprinting mechanism unregulated, leading to different methylation statuses. A series of genes that should exhibit monoallelic paternal expression gets methylated and silenced.  On the maternal allele of chromosome 15, there is the Ubiquitin protein ligase E3A (UBE3A) gene that should exhibit tissue-specific monoallelic expression. Similarly, due to a deletion in the ICR, the imprinting mechanism becomes unregulated, leading to the suppression of the UBE3A gene. What sets this gene apart from other imprinted genes is its tissue-specific imprinting, specifically occurring in brain tissues. Only the maternal allele is expressed in brain cells. Disruption of the imprinting mechanism for this gene results in the lack of expression from both alleles in brain cells, leading to Angelman syndrome. Prader-Willi Syndrome: Characterized by developmental delay, intellectual disability, obesity, short stature, hypogonadism, and dysmorphic features. It's the most common cause of syndromic obesity. Hands and feet are typically small. Angelman Syndrome: Characterized by developmental delay, movement and balance disorders, a distinct gait with wide-based stance, tremulousness in the legs, and uncoordinated movements. Small head circumference. Abnormal EEG (Electroencephalography). Persistent happy demeanor. Hypermotor behavior, short attention span Rubinstein-Taybi Syndrome Rubinstein-Taybi Syndrome is an autosomal dominant inherited syndrome characterized by broad thumbs and toes, short stature, cognitive impairment, and anomalies affecting various systems. Among the molecular causes of this syndrome are deletions in the 16p13.3 region, as well as mutations in the CREBBP(cAMP-response element-binding protein) and EP300 (E1A Binding Protein) genes. The pathogenesis of Rubinstein-Taybi Syndrome is linked to alterations in the epigenetic histone code due to dysfunction in the CBP histone acetyltransferase This condition leads to disrupted gene regulation. While this syndrome is commonly recognized by its physical characteristics, molecular-level changes are one of the underlying reasons for this condition. Fragile X Syndrome (Triplet Repeat Disorders): Errors in the DNA methylation mechanism are associated with certain repeat disorders. The repeat sequences in the genome should exist in a specific number. An increase in the number of repeats beyond the normal count leads to several pathological conditions. Some repeat sequences with a high CG content increase their potential for methylation. Regions that typically contain a certain amount of repeats, when increased in some way, become targets for methylation.  The fragile X mental retardation 1 (FMR1) gene, responsible for mRNA transport, undergoes alterations when the CGG repeats in its 5’UTR region exceed 200 copies. The excessive number of CGG repeats is believed to lead to abnormal methylation conditions. Research has shown that patients exhibit de novo methylation in the elongated CGG repeats in the 5’UTR region and methylation in the promoter region  Individuals with Fragile X Syndrome exhibit certain intellectual, behavioral, and physical differences. This syndrome can affect both sexes. Physical Features: Broad forehead and jaw, Long and narrow face, Large and prominent ears and hands, Strabismus, High-arched palate, Loose joints, Muscle laxity, Flat feet. Behavioral Features: Attention-deficit hyperactivity, Hypersensitivity to touch, Avoidance of eye contact, Irritability, aggression, Repetitive behaviors, Signs of autistic behavior. Cognitive Features: Problems with language and speech, Speech delay, Word repetition and spelling problems, Difficulty in fine and gross motor skills, Difficulty in perceiving emotional cues and providing appropriate responses.