Epigenetics Flashcards
What are epigenetics marks?
Epigenetic marks are changes to the gene expression that don’t change the gene sequence or their coding function. Epigenetic marks are inherited.
Why is folate important in DNA methylation?
Folate produces methionine during metabolism which will provide methyl donors required for DNA methylation. S-adenosyl methionine (SAM) is the principal methyl donor that is catalysed by DNA methyltransferases (DNMTs) to donate their methyl groups to cytosine at CpG dinucleotide sites to form 5-methylcytosine and become S-adenosyl homocysteine (SAH). This will modify cytosine without altering its coding function.
What are the types of DNA methyltransferases (DNMTs)?
De Novo (DNMT 3A & 3B) and maintenance (DNMT 1) methyltransferases. DNMT 3A & 3B will methylate cytosines at the CpG dinucleotide sites to 5-methylcytosines in un-methylated DNA. During replication, DNMT 1 will conduct methylation maintenance by copying the methylation pattern to the daughter strand of the hemimethylated DNA.
What are the 2 types of chromatin?
Euchromatin has an open configuration as it is decondensed and is associated with being transcriptionally active. Whereas, heterochromatin has a closed configuration as it is condensed and is associated with being transcriptionally inactive. The chromatin structural subunit is the nucleosome where DNA is wrapped around a histone octamer consisting of 2 copies of H3, H4, H2A, H2B.
How does lysine acetylation in histones affect DNA?
Acetylated lysine by HATs has a neutral charge and the chromatins are decondensed with an open configuration (euchromatin) compared to deacetylated lysine by HDACs which are positively charged and chromatins are condensed with a closed configuration (heterochromatin). Hence, deacetylated lysine has strong electrostatic attractions with negatively charged DNA as the histones in the nucleosomes are pulled closer to DNA and the heterochromatin will block the transcriptional machinery’s access to DNA. Hence, transcription is repressed and gene expression is decreased.
How does transcriptional repression occur?
Before DNA replication, the de novo DNA methyltransferases (DNMT 3A/3B) will methylate cytosine to 5-methylcytosines at CpG dinucleotide sites in unmethylated DNA. Then, MeCP2 (methyl-CpG binding protein 2) will bind to the methylated DNA and recruit HDAC1 to cause histone deacetylation which facilitates gene repression. During DNA replication, MeCP2 is still bound to the methylated DNA strand so it recruits DNMT1 to conduct maintenance methylation by copying the methylation pattern onto the daughter strand of the hemimethylated DNA. Then, MeCP2 also recruits HDAC1 which will condense chromatin due to the strong electrostatic attractions of positively charged lysine residues in histone (as a result of deacetylation) and negatively charged DNA. Therefore, the transcriptional machinery cannot access the DNA for transcription to occur and gene expression is repressed.
How is lysine methylated?
Lysine is methylated by lysine methyltransferases (KMTs) which can add up to 3 methyl groups.
what post-TRANSLATIONAL modifications can lysine residues undergo which constitute the “histone code” for the regulation of gene expression?
Methylation, sumoylation, ubiquitination.
How can histone be modified to affect gene expression?
Acetylation and phosphorylation ACTIVATE genes. Methylation and ubiquitination ACTIVATE or REPRESS genes.
hypo-methylation = less MeCP2 is recruited = less HDAC is recruited = hyper-acetylation = open chromatin configuration allows transcription machinery to access DNA = gene expression = activation of proto-oncogenes
hyper-methylation = MeCP2 binds to methylated DNA = HDAC is recruited by MeCP2 = hypo-acetylation = condensed chromatin = transcription machinery cannot access DNA = transcriptional repression = silencing of tumour suppressor genes
What are cytidine analogues and how do they treat cancer?
Cytidine analogues are hypomethylating agents that will bind to DNMTs and inhibit their activity on specific tumour suppressor genes (TSG) to increase their gene expression.
What is genomic imprinting?
Genomic imprinting is characterized by MONO-ALLELIC EXPRESSION where 1 parental allele is silenced (imprinted) through METHYLATION and is not expressed. This affects hundreds of genes associated with diseases as the MUTANT ALLELES can behave as both recessive and dominant.
Paternally imprinted genes (Prader-Willi syndrome) promote GROWTH while the maternally imprinted genes (Angelman/happy puppet syndrome) SUPPRESS growth. Both are caused by a DELETION in the same region of chromosome 5.
What methods can be used to measure epigenetic marks?
Methylation-Sensitive Restriction Enzyme Sequencing (MRE-Seq).
Cytosine Extension Assay to evaluate global methylation.
Methylated DNA Immunoprecipitation (MeDIP).
Bisulfite modification of DNA.
Methylation-specific primers (MSP).
Chromatin immunoprecipitation (ChIP) assay to assess histone modifications.
Whole genome sequencing.
How does Cytosine Extension Assay measure epigenetic marks by evaluating global methylation?
Cytosine extension assay (CEA) uses methylation-sensitive restriction enzymes (MRE) for fragmentation, which can differentiate between methylated and unmethylated DNA. For instance, HpaII will only cut unmethylated DNA as it is blocked by methylation and its counterpart MspI will cut both methylated and unmethylated DNA. A Taq polymerase will then extend the fragment overhang and tag the unmethylated DNA with a radiolabelled cytosine, this allows the quantification of methylation. Fragment size analysis will then identify the methylation sites within the gene sequence as the DNA cut with HpaII will produce longer sequences due to uncut methylated DNA.
What are the advantages and disadvantages of the cytosine extension assay?
The cytosine extension assay (CEA) can quantify methylation across the genome, estimate the overall methylation between groups, and is a cheap method to perform. However, the CEA is limited to the CpGs in the HpaII sites, cannot locate the genome methylation differences, is a time-consuming method to perform, and also uses radiolabelled cytosine.
How does Methylated DNA Immunoprecipitation (MeDIP) measure epigenetic marks using SEQUENCING or a DNA MICROARRAY CHIP?
Methylated DNA immunoprecipitation (MeDIP) will fragment DNA using sonication & heat denaturation. Then, it immunoprecipitates the methylated fragments with either a 5-methylcytidine antibody to sequence with MeDIP-SEQ for methylation analysis or immunoprecipitates the methylated fragments with a Cy5-label to hybridize them to an array/CpG island for the comparison with the input DNA on a DNA microarray chip (MeDIP-CHIP) for methylation analysis.
