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Trends Biomedical Science

04c Finding Epigenetics Changes – stimulus questions

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We can treat DNA with sodium bisulfite.Unmethylated cytosine is changed to uracil,

while methylated cytosine is unaffected.

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How can we use this to find methylated DNA?

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What will happen to the these DNA sequences when we treat them with bisulfite?

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What would happen if you PCR amplified the DNA?

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What would happen if you PCR amplified the DNA? Cytosine residues at originally methylated positions, and thymine residues at originally unmethylated position (that were converted to uracil).

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What would we have to think about when we chose the primers for the PCR?

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What would we have to think about when we chose the primers for the PCR?

Primers used during this step do not contain CpG sites (the common target of cytosine methylation), so the amplification works for both methylated and unmethylated DNA.

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How could we use restriction enzymes to tell us about the methylation?

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How could we use restriction enzymes to tell us about the methylation?

PCR products are treated with a restriction enzyme (e.g. BstUI), which will only cut sites that were originally methylated (CGCG), while leaving sites that were originally unmethylated (TGTG).

12https://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis#/media/File:Cobra_workflow.svg

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QuantificationThe digested fragments are then separated

by polyacrylamide gel electrophoresis with the expected appearance of bands corresponding to a single large undigested fragment, and many smaller bands corresponding to digested fragments.

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Quantitative amount of DNA in these bands can be measured.

The methylation percentage of the original sample can be calculated by:

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This technique is called Combined Bisulfite Restriction Analysis (or COBRA)

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UsesCOBRA has been in many applications such as:• screening for DNA methylation changes

at gene promoters in cancer studies, • detecting altered methylation patterns at

imprinted genes, and • characterizing methylation patterns in the

genome during development in mammals.

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In medicine, COBRA canhelp diagnose human disease involving incorrect DNA methylation. COBRA is used in the diagnosis of the genetic imprinting disorder Russell-Silver syndrome where hypomethylation of the imprinted gene H19 is responsible for the disorder in up to 50% of patients.

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What other ways could we use the bisulfite technique to find methylated sites?

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Non-methylation-specific PCR based methods• Direct sequencing• Pyrosequencing• Methylation-sensitive single-strand

conformation analysis (MS-SSCA)• High resolution melting analysis (HRM)• Methylation-sensitive single-nucleotide

primer extension (MS-SnuPE)• Base-specific cleavage/MALDI-TOF

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Methylation-specific PCR (MSP)

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Immunological techniques.Especially immunoprecipitation

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Methylated DNA immunoprecipitation (MeDIP or mDIP) is a large-scale (chromosome- or genome-wide) purification technique in molecular biology that is used to enrich for methylated DNA sequences.

An antibody raised against 5-methylcytosine (5mC) is used to enrich methylated DNA fragments .

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Can you explain what happens here?

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Applications of MeDIPInactive X-chromosome in females is hypermethylated on

a chromosome wide level (MeDIP coupled with microarray).

Colon and prostate cancer cells using MeDIP-chip. Gives genome-wide analysis of genes lying in hypermethylated regions – there is de novo methylation in cancer cells.

High resolution methylome mapping in Arabidopsis using MeDIP-chip.

MeDIP-chip to investigate human breast cancer for methylation associated silencing. Found the inactivation of the HOXA gene cluster.

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Restriction Landmark Genomic Scanning (or RLGS)

Lets us quickly see thousands of restriction sites. Using a combination of restriction enzymes

some of which are specific to DNA modifications, the technique can be used to see differences in methylation levels across the genome of a given organism.

RLGS can be used to find the methylation of thousands of CpG islands—simultaneously.

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RLGS becomes very useful when doing whole-genome scans, and can do the work of thousands of polymerase chain reactions at once.

It easily finds changes from normal, so is very effective in finding hyper/hypomethylation in tumors, deletions or amplifications of genes, or changes in gene expression throughout the development of an organism.

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Chromatin immunoprecipitation (ChIP)• used to find DNA binding sites on

the genome for a particular protein of interest.

• gives a picture of the protein–DNA interactions that occur inside the nucleus of living cells or tissues.

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35https://en.wikipedia.org/wiki/Chromatin_immunoprecipitation#/media/File:Chromatin_immunoprecipitation_sequencing.svg

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DNA-binding proteins (including transcription factors and histones) in living cells can be cross-linked to the DNA that they are binding.

By using an antibody that is specific to a DNA binding protein, we can immunoprecipitate the protein–DNA complex out of cellular lysates.

The crosslinking is often done by applying formaldehyde to the cells (or tissue).

Cells are then lysed and the DNA is broken into pieces 0.2–1.0 kb in length by sonication.

Immunoprecipitation purifies protein–DNA complexes. The purified protein–DNA complexes are then heated to

reverse the formaldehyde cross-linking , allowing the DNA to be separated from the proteins.

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The identity and quantity of the DNA can then be found by PCR.

We must have an idea which genomic region is being targeted in order to generate the correct PCR primers.

We could get around this by cloning the isolated genomic DNA into a plasmid vector and then using primers that are specific to the cloning region of that vector.

To find where the protein binds on a genome-wide scale, a DNA microarray can be used (ChIP-on-chip or ChIP-chip).

ChIP-Sequencing can localize protein binding sites in a high-throughput, cost-effective fashion.

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