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DNA Methylation Influence on Transcription at Promoter

-methylation of CpG islands within core promoters (in most cases) results in transcriptional inhibition -histone methylation somewhat more complicated

-transcription can either be repressed or activated depending on histone and position of amino acid that is being methylated as well as level of methylation

-methylated state of CpG island can have profound impact on histone modifications and vice versa

-some cases, methylation of one is dependent upon methylation of the other (self-reinforcing loop)


Histone Modifications Influence on Transcription at Promoter

-histone modifications such as acetylation and phosphorylation also influence transcription

-in general these two marks will promote transcription by making DNA more accessible to transcription factors and RNA Pol II.


Activation/Repressing Histone Modification

-addition of acetyl groups to the N-terminal tails of histone core proteins serves to activate transcription

-added by a set of enzymes called Histone Acetyl Transferases (HAT)

-human CREB Binding Protein (CBP) contains a HAT domain and that mutations in CBP lead to Rubinstein-Taybi Syndrome

-addition of phosphoryl groups to histone core proteins is another activating modification.

-additions usually occur at either serine or threonine amino acids within the N-terminal tails.

-2 types of proteins mediate these modifications: kinases and Histone Phosphoryl Transferases (HPT)


Histone Methylation Locations (Activation/Repression)

-can be methylated at two different amino acids: arginine and lysine.

-arginine amino acids are either monomethylated or dimethylated

-lysine residues can be monomethylated, dimethylated or trimethylated

-whether a gene transcriptionally activated or silenced is dependent upon the level of methylation and the position of the lysine or arginine residue within the histone N-terminal tail

-identity of the histone (H3 vs H4) is also important in determining how transcription will be affected


Meditation of Methyl Group Addition to Histones

-addition of methyl groups to histones is mediated by Histone Methyl Transferases (HMTs).

-some are called RMT and others are called LMT based on the amino acid specificity of the enzyme


The Histone Code

-all four histone core proteins (H2A, H2B, H3 and H4) are modified by enzymes that add phosphate, acetyl and methyl groups.

-occur at very specific amino acid residues within the N-terminal tails.

-ex: histone H2A is phosphorylated at Ser-1 but not at Ser-16, Arg-17, Ser-18, Ser-19 or Ser-20

-individual histone core proteins can be simultaneously modified at multiple amino acids

-similarly, within a single nucleosome each of the eight histone proteins can be simultaneously modified

-transcriptional activation or repression depends upon the type of modification, the level of modification, the histone subunit and the amino acid in question.

-this combinatorial code of modification is referred to as the histone code


Methylation of Lysine Residues

-methylation of some lysine residues are always associated with transcriptional activation while methylation of other lysine amino acids are only associated with transcriptional repression

-are situations in which monomethylation is associated with activation while dimethylation or trimethylation of the same lysine amino acid serves as a switch to repressive activity


Code of Histone Methylation and Transcription (Know trends of graphic)

-most extensively studied modification is histone H3 methylation.

-H3 amino acids that are known to be methylated are H3K4, H3K9, H3K27 H3K36, and H3K79.

-each of these lysine amino acids can be either monomethylated, dimethylated or trimethylated

-these modifications or “histone marks” are associated with either transcriptional activation and repression

-H3K4 methylation is always associated with transcriptional activation irrespective of the whether H3K4 is monomethylated, dimethylated or trimethylated

-the other amino acids are associated with both activation and repression depending on the level of methylation.


Methylation Marks Influence on Transcription

-core promoters of eukaryotic genes are bound by histones

-if these histones are modified by activating marks then the core promoter is made accessible to the general transcription factors and RNA Pol II

-if, on the other hand, the histones at the core promoter are modified with repressive marks then the core promoter is made inaccessible to these transcription enzymes


DNA Methylation of CpG Islands within Core Promoter

-region surrounding the transcriptional start site is enriched in CG repeats (up to several thousand)

-since the concentration of the CG repeats at the core promoter are significantly higher than the rest of the genome these are called CpG islands

-if these islands are unmethylated then the core promoter remains open and accessible to the general transcription factor machinery

-if CpG islands are methylated then this leads to the deacetylation of histones by HDACs and the trimethylation of H3K27

-these two histone modifications lead to a closing of the core promoter and the prevention of binding by the general transcription factors and RNA Pol II

-this results suggests that there is communication between enzymes that methylate DNA and enzymes the methylate histones

-methylation of DNA occurs at cytosine nucleotides (within the CpG island) and is mediated by DNA methyl transferases (DNMT)


DNA and HIstone Metylation Cross-Talk (KDMA)

-CpG islands that are unmethylated are associated with nucleosomes in which H3K27 methylation (repressive mark) is absent while retaining the H3K4 methylation (activating mark)

-link between DNA and histone methylation and how is H3K27 trimethylation mark removed?

-binding protein KDMA within in ZF-CxxC DNA is recruited to unmethylated CpG islands within the core promoter through its specialized zinc finger domain

-also contains a H3K27 demethylase which means that it specifically removes methyl groups for H3K27.

-this molecule therefore links the processes of DNA and histone methylation

-Since the ZF-CxxC motif binds specifically to unmethylated CpG sequences this allows for the specific removal of repressive histone marks at promoters that need to be activated by RNA Pol II

-in contrast, promoters that contain methylated CpG islands do not recruit KDMA.

-so, repressive H3K27 histone mark remains at the core promoter of genes that are required to be inactive


BiSulfite Sequencing Determination of Methylated Cytosine Nucleotides

-bisulfite sequencing is a method by which one can determine which cytosine bases within the genome are methylated.

-can help to identify genes that are transcriptionally active (unmethylated CpG island) and those that are repressed (methylated CpG island).

-treatment of DNA with bisulfite leads to the deamination of unmethylated cytosine nucleotides into uracil nucleotides.

-in contrast methylated cytosine nucleotides are unaffected by bisulfite treatment

-in order to determine which cytosine nucleotides are methylated in the genome one can treat genomic DNA with bisulfite and then sequence the genomic DNA.

-any residue that is read by the sequencing machine as a cytosine was methylated and any residue that is read as a uracil was a cytosine that was not methylated.

-note that 5methyl cytosine can be deaminated by cellular enzymes to yield a thymine nucleotide but bisulfite treatment has no effect on this residue