MARCO - DNA methylation & id Flashcards
Gene expression can be controlled by
- DNA methylation
- Histone modifications
o Histones methylation o Histone acetylation
These are considered epigenetic changes - affect the availability of DNA/ chromatin for binding of Transcription Factors - Long non-coding RNA (lncRNA) can also facilitate control of gene expression by promoting/ preventing DNA/ histone methylation
- miRNA/siRNA – degrades mRNA/ disrupt translation (in Gilestro L3)
DNA methylation:
Reversible addition of methyl group (CH3) to C5 of cytosine sugar group
* Deposited by methyltransferases (DNMT3a & DNMT3b in mammals)
DNA methylation is a repressive form of control of gene expression. DNA methylation forms compact, condensed chromatin which cannot be accessed by transcription factors, RNA polymerase, & other co-factors
Conservation of DNA methylation:
Methylation is maintained through cell division
* During mitosis, due to DNA replication, the copied DNA strand will be unmethylated, resulting in hemi-methylated DNA
* Hemi-methylated DNA is recognized by DMNT1, which methylates the new strand to maintain the methylation state.
Location of methylation
- Intergenic regions
- Repetitive elements (transposons):
- CpG islands
- Intergenic regions
(regions in between genes - introns)
* usually methylated to maintain chromatin stability/ integrity by:
o forming compacted chromatin which is less accessible for recombination and translocation
o silencing aberrant splice sites that could corrupt the transcript
o silencing aberrant promotors
§ ex. the existence of promotor in the intergenic region on the opposite strand could result in the collision of RNA pol 2 during transcription, causing transcriptional interference
- Repetitive elements (transposons):
- Transposons = repetitive DNA sequences that have the capability to move (transpose) from one location to another in genome, introducing mutations and disruptions to specific genomic regions
- Usually methylated, both at DNA & histones level, to restrict their movement and disruptive potential by:
o silencing incorrect promotors in the repeats, preventing incorrect transcription of downstream gene
o causing mutation of methylated C to T over evolutionary time, thus manipulating the repetitive sequence. The mutation could end up preventing its transposition/ recombination with other regions.
§ Sequences of transposons code for transposases which allows the transposons to be cut and copied to a different site.
§ Mutation in those sequences result in loss of transposase function, preventing transposition of transposons.
§ 50% of human genome is made of transposons, but 99% of those transposons have lost their function due to accumulated mutation.
- CpG islands
- region of genome rich in CG content located upstream of genes, near promotors & enhancers
- Usually unmethylated to allow for transcription factor recognition of promotors
- Methylation of CpG islands will result in silencing of gene expression due to TF being unable to bind promotors. Over time, methylation can result in mutation of C to T, which will end up permanently disrupting the promotor & preventing TF recognition & expression of the downstream gene.
DNA methylation & diseases/cancer
- DNA methylation patterns in disease tissues are different from those in normal tissue
- Abnormal methylation of certain genes has been correlated with cancer and
neurodegeneration.
o Ex. Alzheimer’s disease (NEP gene), Colorectal cancer (MGMT gene), Breast cancer (PRLR gene)
o Could be due to deficiency of DMNT1
o This is because abnormal methylation results in aberrant activation of specific genomic sites, silencing of tumor suppressor genes, eliciting chromosome instability
o Observing abnormal methylation patterns can aid in identification of disease- causing genes
Methylation is important in::
- Gene regulation - silencing gene expression by controlling transcription factors’ access to promotors (@CpG islands)
- Maintaining genomic integrity by forming compact chromatin, silencing aberrant splice sites and promotors (@intergenic regions)
- Silencing transposons (at their promotors or within sequence) (@repetitive sequences)
- Hypomethylation is widely associated with cancer – therefore, it is important to quantify levels of methylation
Identifying DNA methylation regions:
- MeDIP-Seq
- Bisulphite sequencing
MeDIP-Seq (method)
Immunoprecipitation with an antibody that is able to recognize methylated cytosine
1. DNA of interest is fragmented by sonification & denatured
2. The methylated & non-methylated DNA are separated by immunoprecipitation using an antibody that binds to the methylated cytosine. Antibody-bound methylated DNA is kept in the precipitate.
3. Isolated fraction (the methylated regions) are sequenced using a next generation sequencing platform
4. Sequences are mapped back onto the genome to identify methylated regions
MeDIP-Seq (normal vs disease samples)
Location of methylated regions are compared between normal and disease samples to identify genes related to those diseases.
* Ex. breast cancer: 80% of breast cancer cells lost do not display methylation present in normal human mammary epithelial cells at the HSATII RNA gene.
Whole-genome DNA methylation profiles for human breast cancer cell lines (BCC) & for normal human mammary epithelial cells (HMEC)
Bisulphite sequencing
- Treat DNA sample with Bisulphite which converts cytosine to uracil, but sample with methylated cytosine is unaffected
- Treated sample are then amplified by PCR twice using diff. primers – non-methylation specific PCR & methylation specific PCR
* unmethylated region of sample will be amplified using primers w/ uracil since all cytosine = turned to uracil in non-methylation specific PCR
* methylated region of sample will be amplified in methylation specific PCR - Sequence the DNA sample
- Map sample sequence with a reference genome & observe alignment
* Region that aligns = methylated since the sequence has not been disrupted by Bisulphite
* Region that does not align = unmethylated since all cytosine = turned to uracil & not identical to reference anymore
Bisulphite sequencing characteristics
- Higher cost but greater resolution
- Can treat on targeted regions or whole genome approach (WGBS)
- Can treat on diff samples and compare alignment ex. cancer vs normal cells – observe the region that differs in methylation – can identify the gene responsible for causing cancer
