Methylation Flashcards
Describe Epigenetic modification and the relevance to human disease, clinical diagnostic testing or gene identification.
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What modifications do histones have?
odifications affect specific amino acids in the tails. INclude acetylation, monomethylation, dimethylation or trimethylation of lysines and phosphorylation of serines. These are effected by large families of enzymes: histone aceyltransferases, histone methyltransferases, histone kinases.
How does histone define the conformation of chromatin?
Paricular combinations of histone modifications ay define its conformation. However no single histone modification is completely predictive of chromatin state or DNA activity. The various histone modifications influence chromatin structure and function by acting as binding sites for a wide range of nonhistone effector proteins. These proteins contain domains that recognise specific histone modifications: bromodomains recognise acetylated lysines and chromodomains recognise lysinces that are methylated.
What are the features of euchromatin?
nucleosomes are loosely packed, with nucleosome-free regions that can bind regulatory proteins.
What are the features of heterochromatin?
Nucelosomes are densely packed and associated with heterchromatin protein 1. Gene-poor and contains much repetitious DNA sequence, and replicates late during S phase.
What techniques are available to study chromatin conformation (ie whole genome methylation)?
ChIP (chromatin immunoprecipitation)
crosslink proteins to DNA, lyse cells and isolate chromatin (fragmented), and an antibody against some protein of interest is used to precipitate the chromatin fragments that inlcude the protine (precipitate antibody-bound protine-DNA complexes). Reverse crosslinking, degrade protein and purify DNA fragments. The recovered DNA can be hybridised to a microary to identify genomewide associations (ChIP-chip), tag sequence fragments ChIP-PET or STAGE or sequence fragments ChIP-seq.
What factors can change the conformation of chromatin?
DNA methylation, histone modification and nucleosome positioning. Histone-modifying enzymes - eg methyltransferases, kinases, acelytransferases previously mentioned.
ATP-dependent remodeling complexes (ATPase and partners) change the nucleosome occupancy of DNA - various families. They move nucleosoes along the DNA or promote nucleosome assembly or disassembly. Both locally and globally, nucleosome positioning affects gene expression.
How does DNA methylation control gene expression?
methylation of CpG acts as a signal that is recognised by specific MeCpG-binding proteins. These have a role in regulating chromatin structure and gene expression. and epigenetic memory. Cytosine methylation is achieved by DNA methyltrasnferases (DNMTs). Humans have 3. A fourth (DNMT3L helps target methylases to appropriate sequences and fifth DNMT2 works on RNA. ~70% CpG methylated concentrated on repetitive sequence (including satellite repeats of pericentric heterchromatin and dispersed transposons. Methylation also sporadically dispersed in itrons and exons and intergenic sequences. The CpG dinucleotide is the least frequent dinucleotide due to tendency of methylated cytosines to deaminate to thymines. CpG islands at promoters represent an exception.
Some TF fail to bind. DNA methylation can also be read by proteins containing a methyl-CpG-binding domain (MBD) which can recruit other proteins associated with repressive structures such as HDACs. Humans have 5 methyl binding proteins - MBD1-4 and MECP2. Mutually reinforcing histone and DNA methylation in heterochromstin - intarct. DNA methyltransferases and MeCpG-binding proteins recruit repressive complexes that contain HDACs and/or histon methyltransferases. Methylated H3K9 can bind chromodomain proteins such as HP1 (small nonhistone protein may mediate spreading of heterochromatin)which can in turn recruit DNA methyltransferases. Mtually reinforcing effects maintain repressive chromatin states.
How does DNA methylation change during development?
Erasure of parental epigenetic settings and gonadal differentiation. De novo methylation and establishment of imprinting marks. Germ cells develop (egg and sperm cells heavily methylated DNA ferrtilised –> oocyte. Genomwide demethylation in early embryo (blastocyst). Then genomewide de novo DNA methylation in blastocyst. The extent of this methylation varies in different cell lineages: somatic cell lineages are heavily methylated - and maintained in the adult. Whereas trophoblast-derived lineages (giving rise to placenta, yolk sac etc) are relatively undermethylated.
What is the role of small RNA molecules in hetrochromatin>
Heterochromatic regions are coated wtih components of the RNAi-induced transcriptional silencing complex. One component of this complex is a chroodomain containing protein which may explainhow the RITS could be targeted to chromatin containing ethylated H3K9. Silencing of heterochromatin requires transcription from repeat elements and these trasncripts are processed to siRNAs. siRNAs can rigger the methylation of histones.
